CN109486902A

CN109486902A - Nucleic acid amplification

Info

Publication number: CN109486902A
Application number: CN201810576963.4A
Authority: CN
Inventors: C-Y·李; D·拉夫; S-M·陈; J·奥尼尔; R·卡辛斯卡斯; J·罗恩伯格
Original assignee: Life Technologies Inc
Current assignee: Life Technologies Inc
Priority date: 2012-04-19
Filing date: 2013-03-15
Publication date: 2019-03-19
Anticipated expiration: 2033-03-15
Also published as: SG10201802883UA; WO2013158313A1; SG11201406717RA; CN104471075A; CN114854832A; CN116064734A; CN104471075B; CN109486902B

Abstract

In some embodiments, the present teachings provide methods for nucleic acid amplification comprising forming a reaction mixture, and subjecting the reaction mixture to conditions suitable for nucleic acid amplification. In some embodiments, methods for nucleic acid amplification include subjecting the nucleic acid to be amplified to partially denaturing conditions. In some embodiments, a method for nucleic acid amplification comprises amplifying without fully denaturing the nucleic acid being amplified. In some embodiments, methods for nucleic acid amplification use enzymes and polymerases that catalyze homologous recombination. In some embodiments, methods for nucleic acid amplification can be performed in a single reaction vessel. In some embodiments, methods for nucleic acid amplification can be performed in a single continuous liquid phase of the reaction mixture without the need for compartmentalization of the reaction mixture or immobilization of reaction components. In some embodiments, the method for nucleic acid amplification comprises amplifying at least one polynucleotide on a surface under isothermal amplification conditions, optionally in the presence of a polymer. The polymer may include a sieving agent and/or a diffusion reducing agent.

Description

Nucleic acid amplification

Background of invention

The application is the divisional application application No. is 201380031868.1.

Nucleic acid amplification is highly useful in molecular biology and actually in biology, acology, diagnostics, medical jurisprudence There is wide applicability with each aspect of research.Normally, expansion is generated from starting template using one or more primers Increase son, wherein amplicon corresponds to or be complementary to the template that the amplicon is generated from it.Multiplex amplification can also make process simplification And reduce expense.The application is related to method and reagent for nucleic acid amplification and/or analysis.

Summary of the invention

Method, reagent and the product of nucleic acid amplification and/or analysis is provided herein.Amplification can using fixed and/or The primer of dissolution.Single group primer can be mixed from different templates, or single template can be contacted with a variety of different primers, Or a variety of different templates can be contacted with a variety of different primers.The amplicon generated from methods provided herein is suitable It shares in the substrate of further analysis such as sequencing.

In some embodiments, this teaching content provides the composition for nucleic acid amplification, system, method, apparatus And kit.

Attached drawing is described in detail

Fig. 1 provides the schematic diagram of the embodiment of indicating template walking (template walking).Selectable In embodiment, fixed primer includes to be named as (A)_nThe sequence rich in adenosine, such as (A)₃₀, and be directed in template The primer binding site of immobilized primer includes the complementary sequence rich in T, such as (T)₃₀。

Fig. 2 describe on globule by template walk amplification and globule planar array on accumulation to be used to survey The sketch plan of sequence.

Fig. 3 describes some selectable embodiments of the detection based on semiconductor using synthesis order-checking.Template row It walks can be used for generate clonal expansion subgroup on globule or in the substrate of reaction chamber or bottom.In selectable embodiment In, fixed primer includes to be named as (A)_nThe sequence rich in adenosine, such as (A)₃₀, and immobilized primer is directed in template Primer binding site include the complementary sequence rich in T, such as (T)₃₀。

Fig. 4 describes some optional of the fixation site in the form of primer lawn (primer lawn) on a planar substrate The embodiment selected.The single continuous lawn of array or primer that discontinuous fixed site can be used can be considered as fixed The random array in site.Optionally, position of one or more fixed sites in the continuous lawn of primer can be also not true Fixed, wherein position is expert at when the attachment of starting template until then and is determined, or space occupied by the cluster by expanding is Lai really It is fixed.In selectable embodiment, fixed primer includes to be named as (A)_nThe sequence rich in adenosine, such as (A)₃₀, It and include the complementary sequence rich in T, such as (T) for the primer binding site of immobilized primer in template₃₀。

Fig. 5 shows influence of the temperature to template walking reaction.Template walking is calculated and depicts for reaction temperature to expand The chart of Δ Ct before and after increasing.

Fig. 6 provides the table of the Ct value of 96 dual TaqMan qPCR reactions.

Fig. 7, which is described, shows the data of the about 100,000 times of amplifications carried out on globule by template walking.For anti- The Δ Ct before and after template walking is reacted and the amplification before and after template walking reaction are calculated and depicted between seasonable Multiple.

Fig. 8 provides exemplary chain overturning and the schematic diagram of Running strategy describes.(A) template is walked, and (B) chain is overturn to produce The chain of raw overturning, (C) adds new primer binding sequence Pg ' on final overturning chain.

Fig. 9 describes the Ion Torrent from the polynucleotide template for using the amplified reaction of recombinase-mediated to expand^TM The exemplary reading length histogram of PGM sequencing operation.

Figure 10 describes the Ion from the polynucleotide template for using the amplified reaction of recombinase-mediated to expand Torrent^TMThe exemplary reading length histogram of Proton sequencing operation.

Figure 11 describes the Ion from the polynucleotide template for using the amplified reaction of recombinase-mediated to expand Torrent^TMThe exemplary reading length histogram of Proton sequencing operation.

Figure 12 describes the Ion from the polynucleotide template for using the amplified reaction of recombinase-mediated to expand Torrent^TMThe exemplary reading length histogram of Proton sequencing operation.

Figure 13 includes the diagram of exemplary measurement system.

Figure 14 includes the diagram of exemplary measurement component.

Figure 15 includes the diagram of the array of exemplary measurement component.

Figure 16 includes the diagram of exemplary bore structure.

Figure 17 includes the diagram of exemplary bore and sensor structure.

Figure 18, Figure 19, Figure 20 and Figure 21 include the diagram of the work package during being handled by illustrative methods.

Figure 22, Figure 23 and Figure 24 include the diagram of the work package during being handled by illustrative methods.

Figure 25, Figure 26 and Figure 27 include the diagram of the work package during being handled by illustrative methods.

Figure 28 shows the exemplary block diagram of the component of the system for nucleic acid sequencing according to exemplary implementation scheme.

Figure 29 shows the exemplary cross of the part of the IC apparatus and flow cell according to exemplary implementation scheme Figure.

Figure 30 is shown according to the exemplary of the representative chemical sensor of exemplary implementation scheme and corresponding reaction zone Sectional view.

Detailed description of the invention

Nucleic acid-templated conventional amplification generally includes the repetition of the template (and/or its filial generation) using synthesis system appropriate Duplication.In such conventional method, each example of duplication is usually denaturalized by using extreme Denaturing wait be amplified Template start so that template is substantially single-stranded.Extreme Denaturing for conventional amplification it is some usually With the thermal denaturation (example that widely used example includes using the temperature much higher than nucleic acid-templated melting temperature to be amplified Such as, Standard PCR includes the normally about thermal cycle of 94-95 DEG C of denaturation temperature using being much higher than 90 DEG C) or template to strength The exposure of denaturant such as NaOH, guanidine salt reagent etc..Such method usually requires special equipment (such as thermal cycler), and And additional operation (such as annealing steps for Standard PCR are needed during amplification procedure；For removing chemical denaturant Washing step etc.), to increase cost relevant to such amplification, workload and time, and limits and use this The yield that the method for sample can be obtained finally.In addition, such extreme Denaturing usually makes template to be amplified be basic It is upper single-stranded, thus to be related to multiple clonal expansion (i.e. clonal expansion of the multiple different templates in identical reaction mixture) Extensive application propose challenge.For such multiple application, the use of these extreme Denaturings, which can be, runs counter to desire , because this typically results in a chain of template from the release of its position of associating, so that the chain of release is freely in solution It is interior migration and pollute it is other develop in close proximity to amplicon.Such cross contamination typically results in reduced monoclonal amplification The yield of group and the yield for increasing polyclonal pollutant (being not generally available to many downstream applications).Improved nucleic acid is needed to expand Increasing method (and relevant composition, system and kit) is to eliminate defect relevant to conventional amplification method.

In some embodiments, present disclosure generally relates to the method and relevant combination of nucleic acid amplification Object, system and device, the method includes amplification of nucleic acid templates to generate the expansion comprising the substantially polynucleotides group of monoclonal Increase son.It has been generally acknowledged that monoclonicity is desired in nucleic acid determination, because the difference of different polynucleotides is special in polyclonal group Property may make the explanation of determination data to complicate.One example is related to nucleic acid sequencing application, wherein the presence of polyclonal group can make The explanation for obtaining sequencing data complicates；However, many sequencing systems, which are not enough to sensitive arrive from single polynucleotide template, detects core Nucleotide sequence data, therefore need before sequencing the clonal expansion of template.

In some embodiments, the amplification method of present disclosure can be used for optionally using identical reaction mixture It is different nucleic acid-templated with two or more are expanded to clone in identical reaction mixture, it is basic to generate at least two The nucleic acid group of upper monoclonal.Optionally, the amplification shape that the group of at least one substantially monoclonal passes through single polynucleotide template At.

Optionally, two or more different nucleic acid-templated simultaneously and/or are in parallel expanded.

In some embodiments, present disclosure generally relates to (and the relevant combination of nucleic acid synthetic method Object, system and kit), which comprises at least two double-stranded nucleic acid templates are provided in the reactive mixture；With according to this Any method clone ground disclosed herein expands at least two double-stranded nucleic acid template, to form at least two substantially Dan Ke Grand nucleic acid group.

In some embodiments, the amplification of clone ground optionally includes to form reaction mixture.Reaction mixture may include Continuous liquid phase.In some embodiments, continuous liquid phase includes single continuous aqueous phase.Liquid phase may include two or more Polynucleotide template, polynucleotide template nucleotide sequence optionally having the same or can have core different from each other Nucleotide sequence.In some embodiments, at least one of the two or more polynucleotide templates may include at least one A at least one other polynucleotide template in reaction mixture is not substantially identical or substantially not complementary nucleic acid Sequence.

In some embodiments, two or more different nucleic acid-templated be confined to before amplification, be placed in or be located at Different sites.

In some embodiments, in the solution, two or more are expanded to clone optionally in single reaction mixture Multiple and different is nucleic acid-templated, and after such clonal expansion, two or more resulting substantially monoclonals Nucleic acid group is then confined to, is placed in or positioned at different sites.

Different sites is optionally the member of site array.Array may include surface (such as flow cell, electronics dress Set, the surface of transistor chip, reaction chamber, slot etc.) on site two-dimensional array or matrix or other mediums (such as solid, Semisolid, liquid, fluid etc.) in site cubical array.

Optionally, two or more different nucleic acid-templated Continuous Liquid Phases in same reaction mixture, normally connect It is amplified in continuous water phase, so that two or more different and substantially monoclonal polynucleotides groups are generated, wherein each A polynucleotides group is generated by being present in the amplification of single polynucleotide template in reaction mixture.

Optionally, Continuous Liquid Phase is included within the single phase or identical phase of reaction mixture.

In some embodiments, present disclosure generally relates to (and the relevant combination of nucleic acid synthetic method Object, system and kit), which comprises double-stranded nucleic acid template is provided；With by expanding the double-stranded nucleic acid template come shape At the nucleic acid group of substantially monoclonal.Optionally, amplification includes that clone ground amplifying doulbe-chain is nucleic acid-templated.

Optionally, amplification, which is included under substantially isothermy, carries out at least one amplification bout.

Optionally, amplification, which is included under substantially isothermy, carries out at least two continuous nucleic acid synthesis circulations.

In some embodiments, amplification includes recombinase polymeric enzymatic amplification (RPA).For example, amplification may include carrying out extremely A few RPA bout.

In some embodiments, amplification includes that template is walked.For example, amplification may include carrying out at least one template walking Bout.

In some embodiments, amplification, which is optionally included in site or reaction chamber, carries out two different expand back It closes.For example, amplification may include carrying out at least one RPA bout simultaneously in site or reaction chamber with any sequence of bout or combination At least one template walking bout is carried out in site or reaction chamber.In some embodiments, any one or more are expanded back At least two continuous circulate under substantially isothermy in conjunction carry out.In some embodiments, bout is expanded at least One carries out under substantially isothermy.

Optionally, it is to be amplified it is nucleic acid-templated be double-strand, or make the mould using program appropriate before amplification Plate is at least partly double-strand.(herein template to be amplified with nucleic acid-templated or polynucleotide template is interchangeable makes With).In some embodiments, template is linear.Alternatively, template can be cricoid, or include linear and annular section Combination.

Optionally, double-stranded nucleic acid template includes positive chain.Double-stranded nucleic acid template also may include reverse strand.Positive chain is optionally Include the first primer binding site.Reverse strand optionally includes the second primer binding site.

In some embodiments, template has included the first and/or second primer binding site.Alternatively, template is optionally Primer binding site is not included initially, primer binding site is connected to or is introduced before being optionally included in amplification by disclosed method Template.For example, method optionally includes being connected to or being otherwise directed into template for the connector comprising primer binding site. Can connector be connected to or is otherwise directed into the end or the linear or main body of circular template of linear die.Optionally, It can circularized template after connecting upper or introducing connector.In some embodiments, can the first connector be connected to or is introduced linearly The first end of template, and can the second connector be connected to or be introduced the second end of template.

In some embodiments, amplification includes by the template of partial denaturation and the first primer, with the second primer or with the One primer and the second primer are contacted with random order or combination.

In some embodiments, the first primer includes the first primer sequence.The first primer optionally includes extendible End (such as 3 ' ends containing OH).The first primer is optionally connected to compound (such as " resistance label (drag Tag it) ") or is connected on support (such as globule or site or surface of reaction chamber).

In some embodiments, the second primer includes the second primer sequence.Second primer optionally includes extendible End (such as 3 ' ends containing OH).The second primer is optionally connected to compound (such as " resistance label ") or is connected to Support (such as globule or site or surface of reaction chamber).

Optionally, the first primer is bound to the first primer binding site to form the first primer-template double-strand.Second primer In combination with to the second primer binding site to form the second primer-template double-strand.

In some embodiments, amplification includes the first primer for extending the first primer to form extension.For example, amplification can Including extending the first primer-template double-strand the first primer to form the first primer of extension.

Optionally, extended through polymerase.Polymerase can be strand displacement polymerase.

In some embodiments, amplification includes contacting template to be amplified with recombinase.

In some embodiments, amplification includes forming the template of partial denaturation.For example, amplification may include that partial denaturation is double Chain is nucleic acid-templated.

Optionally, partial denaturation includes that double-stranded nucleic acid template is made to undergo partial denaturation condition.

In some embodiments, partial denaturation condition includes the temperature less than nucleic acid-templated Tm, including such as less than 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C of Tm nucleic acid-templated of temperature.In some embodiments, partial denaturation item Part includes being higher than (for example, at least 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C are higher) the first primer, the second primer or first With the temperature of the Tm of the second primer.In some embodiments, partial denaturation condition include be higher than (for example, at least 5 DEG C, 10 DEG C, It is 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C higher) the first primer binding site, the second primer binding site or the first primer binding site With the temperature of the Tm of the second primer binding site.In some embodiments, nucleic acid-templated to include in one or two end Joint sequence, and partial denaturation condition may include the temperature of the Tm higher than joint sequence.In some embodiments, part becomes Property condition (particularly partial denaturation temperature) to be used for the selective amplification during " template walking " nucleic acid-templated, it is such as herein It further describes.

In other embodiments, partial denaturation condition include with it is one or more can optionally with sequence-specific or To be amplified nucleic acid-templated of the nucleic acid-templated enzymatic treatment of sequence oriented approach partial denaturation makes its contact.In some embodiment party In case, at least one enzyme optionally with sequence-specific fashion catalysis chain intrusion and/or untwists.Optionally, one or more enzymes Including one or more enzymes selected from recombinase, topoisomerase and unwindase.In some embodiments, partial denaturation template It may include that template is contacted to recombinase and formed the nucleoprotein complex comprising recombinase.Optionally, there are the first primer, Template is contacted with recombinase in the case where second primer or the first and second primers.Partial denaturation may include being urged using recombinase Change chain exchange and hybridizes the first primer with the first primer binding site (or the second primer and the second primer binding site is miscellaneous It hands over).In some embodiments, partial denaturation includes chain exchange being carried out using recombinase and by the first primer and the first primer knot It closes position and hybridizes the second primer with the second primer binding site.

In some embodiments, the template of partial denaturation includes single stranded portion and double stranded section.In some embodiments In, single stranded portion includes the first primer binding site.In some embodiments, single stranded portion includes the second primer engaging portion Position.In some embodiments, single stranded portion includes the first primer binding site and the second primer binding site.

In some embodiments, partial denaturation template includes contacting template with one or more nucleoprotein complexs. At least one of nucleoprotein complex may include recombinase.At least one of nucleoprotein complex may include primer (for example, first Primer or the second primer, or comprising with the primer of the sequence of primer binding sequence complementation corresponding in template).In some embodiment party In case, partial denaturation template may include contacting template with the nucleoprotein complex comprising primer.Partial denaturation may include by core The primer of albumen composition hybridizes with primer binding site corresponding in template, to form primer-template double-strand.

In some embodiments, partial denaturation template may include answering template and the first nucleoprotein comprising the first primer Close object contact.Partial denaturation may include by the first primer binding site of the first primer of the first nucleoprotein complex and positive chain Hybridization, to form the first primer-template double-strand.

In some embodiments, partial denaturation template may include answering template and the second nucleoprotein comprising the second primer Close object contact.Partial denaturation may include the second primer binding site by the second primer of the second nucleoprotein complex and reverse strand Hybridization, to form the second primer-template double-strand.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one Or multiple primer extension procedures.For example, method may include being incorporated to nucleotide by using polymerase to carry out extension primer.

In some embodiments, polymerase is strand displacement polymerase.

Optionally, extension primer includes being incorporated to primer in nucleotide with polymerase and the nucleotide of one or more types Under the conditions of contacted.In some embodiments, the nucleotide of one or more types does not include exogenous marker, particularly light Detectable label on, such as fluorescence part or dyestuff.Optionally, reaction mixture includes nucleotide, is naturally occurring Nucleotide.Optionally, nucleotide does not include the group (such as double deoxidation group, reversible terminator etc.) for terminating nucleic acid synthesis. Normally, extension primer is occurred with template dependent manner.

Optionally, disclosed method (and relevant composition, system and kit) includes by using polymerase by one A or multiple nucleotide are incorporated to the first primer-template double-strand the first primer to extend the first primer, to form the of extension One primer.

Optionally, disclosed method (and relevant composition, system and kit) includes passing through any method appropriate Second primer is bound to the second primer binding site of the primer of the first extension by (such as connection or hybridization).

Optionally, disclosed method (and relevant composition, system and kit) includes by using polymerase by one A or multiple nucleotide are incorporated to the second primer of the second primer-template double-strand to extend the second primer, to form the of extension Two primers.

In some embodiments, extend the formation that the first primer leads to the primer of the first extension.First primer extended It may include some or all of the reversed chain-ordering of template.Optionally, the first primer extended includes the second primer binding site.

In some embodiments, extend the formation that the second primer leads to the primer of the second extension.Second primer extended It may include some or all of the positive chain-ordering of template.Optionally, the second primer extended includes the first primer binding site.

In some embodiments, in the case where not making double-stranded nucleic acid template undergo extreme Denaturing during amplification Carry out method.For example, can be in the case where not making the temperature of the nucleic acid-templated Tm that experience is greater than or equal to template during amplification Carry out method.In some embodiments, can not make template during amplification with chemical denaturant such as NaOH, urea, guanidine Salt etc. carries out method in the case where contacting.In some embodiments, amplification includes isothermal duplication.

In some embodiments, do not make it is nucleic acid-templated at least two, three, four or continuous more than four Method is carried out in the case where undergoing extreme Denaturing during nucleic acid synthesis circulation.For example, method may include two, three, four It is a or synthesize circulation more than four continuous nucleic acid and nucleic acid-templated contacted with chemical denaturant without making.In some embodiments In, method may include progress two, three, four or the continuous nucleic acid synthesis circulation more than four without making nucleic acid-templated warp Go through the reality higher than template or template group or the Tm being computed (template or template group reality or the average Tm that is computed) it Lower 25,20,15,10,5,2 or 1 DEG C of temperature.Two, three, four or more than four continuous nucleic acid synthesis circulation can wrap Include the partial denaturation and/or primer extension procedures of intervention therebetween.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one The primer strand of a or multiple extensions is connected to support.It is carried out optionally during amplification or selectively after the completion of amplification Connection.In some embodiments, support includes multiple second primers, and method may include extend at least one the Second primer hybridization of one primer strand and support.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one Second primer strand of a or multiple extensions is connected to support.In some embodiments, support is connected to the first primer.Example Such as, support may include multiple the first primers, and method may include the second primer and support for extending at least one The first primer hybridization, so that the second primer of extension is connected to support.For example, the first primer can be hybridized to the second of extension The first primer binding site in primer.

In some embodiments, support is connected to the second primer.For example, support may include multiple second primers, And method may include the second primer hybridization of the first primer and support that extend at least one, thus by the first of extension Primer is connected to support.For example, the first primer can be hybridized to the second primer binding site in the first primer of extension.

In some embodiments, support includes at least one the first primer and at least one second primer, and public The method (and relevant composition, system with kit) opened includes connecting the second primer of the first primer extended and extension To support.

Optionally, support is connected to target specific primer.Target specific primer optionally hybridizes (or can hybridize) extremely First subgroup of reaction mixture inner template, but the second subgroup of reaction mixture inner template cannot be bound to.

Optionally, support is connected to universal primer.Universal primer optionally hybridizes (or can hybridize) to reaction mixing All or substantially all templates in object.

Optionally, reaction mixture includes the first support and covalently for being covalently coupled to the first target specific primer It is connected to the second support of the second target specific primer, and wherein the first and second target specific primers are different from each other.

Optionally, the first target specific primer is essentially complementary the first target nucleic acid sequence and the second target specific primer It is essentially complementary the second target nucleic acid sequence, and wherein the first and second target nucleic acid sequences are different.

In some embodiments, disclosed method includes optionally in the identical continuous phase of reaction mixture, The first amplicon is formed by the first template of amplification on one support, and is formed on the second support by expanding the second template Second amplicon.First amplicon optionally connects or is attached to the first support, and the second amplicon optionally connect or It is attached to the second support.

Disclosed method, which is optionally included, by cloning expands two or more polynucleotide templates to generate two Or more monoclonal or substantially monoclonal amplicon.Clone ground optionally in the Continuous Liquid Phase of amplification reaction mixture Expand two or more polynucleotide templates.The Continuous Liquid Phase of amplification reaction mixture may include continuous aqueous phase.In some realities It applies in scheme, amplification includes the polynucleotides group of the substantially amplification of monoclonal of generation at least two, the polynucleotides group's Each is formed by the amplification of single polynucleotide template.Optionally, the amplification of clone ground includes at least one RPA bout. Optionally, the amplification of clone ground includes at least one template walking bout.

In some embodiments, amplification optionally includes to form the amplification reaction mixture comprising Continuous Liquid Phase.One In a little embodiments, Continuous Liquid Phase is single continuous aqueous phase.Liquid phase may include two or more polynucleotide templates, described Polynucleotide template is optionally different from each other.For example, two or more polynucleotide templates may include at least one and expansion At least one other polynucleotide template in increasing reaction mixture is not substantially identical or substantially not complementary nucleic acid sequence Column.

In some embodiments, amplification optionally includes to be formed comprising having two or more polynucleotide templates The amplification reaction mixture of single continuous aqueous phase.Amplification, which is optionally included, by cloning expands two in single water phase or more Multiple polynucleotide templates form the nucleic acid group of two or more substantially monoclonals.Optionally, clone ground, which expands, includes At least one RPA bout.Optionally, the amplification of clone ground includes at least one template walking bout.

In some embodiments, present disclosure generally relates to optionally expand in parallel using partial denaturation condition One or more nucleic acid-templated methods (and relatively composition, system and kit).In some embodiments, optionally Two or more templates are expanded using such method in the form of an array.Optionally, molten before distribution is into array Large quantities of amplification templates in liquid.Alternatively, template is distributed to the site into array first, then at the site of array (within or) Amplification template in situ.

Optionally, template is single-stranded or double-strand.Template optionally includes one or more primer binding sites.

In some embodiments, method may include the double-strandednucleic acid made at least one chain comprising primer binding site Template undergoes at least one to use the replication cycle based on template of polymerase.

Optionally, at least one replication cycle based on template includes partial denaturation step, annealing steps and extension step.

In some embodiments, method includes that continuously the duplication based on template follows by making template experience at least two It is nucleic acid-templated that ring carrys out amplifying doulbe-chain.

In some embodiments, method includes partial denaturation template.Optionally, method includes being formed comprising single stranded zone Partial denaturation template.Partial denaturation template also may include double stranded region.Single stranded zone may include primer binding site.

Optionally, partial denaturation includes that template is made to undergo lower than the Tm of primer binding site at least 20,15,10,5,2 or 1 DEG C temperature.

Optionally, partial denaturation includes that template is made to undergo temperature identical with the Tm of primer binding site or higher than the Tm Degree.

Optionally, partial denaturation includes contacting double-stranded template with recombinase and primer.Recombinase and primer can form core The part of albumen composition, and partial denaturation includes by template and complex contacts.

In some embodiments, method includes being formed and being drawn by hybridizing primer with the primer binding site of single stranded zone Object-template double-strand.In some embodiments, template pending includes double stranded region.Optionally, double stranded region is combined not comprising primer Position.

In some embodiments, method includes extension primer-template double-strand primer.Optionally, method includes being formed The primer of extension.

It in some embodiments, can the clone ground amplification on different discontinuous supports (flowing into globule or particle) Different templates before amplification without being divided.In other embodiments, before amplification by template subregion or distribution extremely In emulsion.Optionally, template is distributed to droplet, forms the hydrophilic of the emulsion with discontinuous aqueous favoring and continuous hydrophobic phase The part of phase.In some embodiments, the emulsion droplets of aqueous favoring also include group necessary to one or more progress PRA Point.For example, emulsion droplets may include recombinase.Optionally, droplet includes strand displacement polymerase.In some embodiments, micro- The drop primer and/or molten phase primers fixed comprising support.Optionally, primer in combination with to template or be bound to its amplification produce Object.

In some embodiments, the amplification disclosed herein for using emulsion-based includes becoming in the part of template Property after carry out composition, system, method, apparatus and the kit of nucleic acid amplification of nucleic acid synthesis and provide compared to Conventional spread The advantageous aspect of method (PCR or emPCR including involving the emulsion-based of conventional thermocy).E.g., including emulsion-based The nucleic acid amplification reaction of the template of RPA (" emRPA ") or emulsion-based walking can produce the polynucleotide template of longer amplification, tool The time for the polynucleotide template for thering is the preparation of less amplification step, reduction to expand and/or increased sequencing data quality.With Such as U.S. Patent number is found in some emulsion compositions appropriate being used together with amplification method disclosed herein 7622280, in 7601499 and 7323305, above-mentioned patent is incorporated herein by reference in their entirety.

In some embodiments, method includes providing comprising the positive chain containing the first primer binding site, containing the The double-stranded template of the reverse strand of two primer binding sites；Partial denaturation template and formation are comprising containing the first primer binding site The partial denaturation template of single stranded zone and at least one double stranded region；By the first primer binding site for making the first primer and single stranded zone Hybridization forms the first primer-template double-strand；Extend the first primer-template double-strand the first primer using polymerase includes to be formed The first primer of the extension of second primer binding site, wherein the first primer extended at least partly hybridizes the forward direction in template Chain；Partial denaturation carrys out the first chain of the extension of self-template to form the single stranded zone comprising the second primer binding site；Second is set to draw Object hybridizes with the second primer binding site of single stranded zone and is formed the second primer-template double-strand, and extends the second primer-template pair Second primer of chain, to form the second primer of extension.

In some embodiments, the method that present disclosure generally relates to the method from nucleic acid-templated nucleic acid (and relevant composition, system and kit), comprising: the first nucleic acid double chain comprising positive chain and reverse strand is provided, wherein Positive chain includes forward primer binding site and reverse strand includes reverse primer binding site, and wherein the first double-strand has First melting temperature (" template Tm "), forward primer binding site have the second melting temperature (" forward primer Tm "), reversely draw Object binding site has third melting temperature (" reverse primer Tm ")；The first double-strand of partial denaturation, the first of part denaturation Double-strand includes the single stranded zone containing forward primer binding site and at least one double stranded region；By making forward primer and partial denaturation The first double-strand forward primer binding site hybridize to form the first double-strand pending；By by the first double-strand pending and chain Metathesis polymerizable enzyme and nucleotide contact under primer extension conditions to extend forward primer, so that being formed has the 4th melting temperature Second double-strand of (" the 4th Tm "), second double-strand include a chain containing forward primer binding site and containing reversely drawing One chain of object binding site；The second double-strand of partial denaturation, the second double-strand of part denaturation include to contain reverse primer knot Close single stranded zone and at least one double stranded region at position；By the reverse primer knot for making the second double-strand of reverse primer and partial denaturation Position hybridization is closed to form reversed second double-strand pending；By by reversed second double-strand and strand displacement polymerase and core pending Thuja acid contacts to extend the reverse primer of reversed second double-strand pending under primer extension conditions.

In some embodiments, method (and relevant composition, system and kit) may also include sequencing amplification Template or sequencing extend primer (such as extension the first primer or extension the second primer).Sequencing may include in this field Any sequencing approach appropriate known.In some embodiments, sequencing includes the sequencing by synthesis or passes through detection of electrons Sequencing (such as nano-pore sequencing).In some embodiments, sequencing include be incorporated to by polymerase-mediated nucleotide come Extend the template of template or amplification or extends the sequencing primer hybridized with the template of template or amplification.In some embodiments, Sequencing include by the primer of template or extension is contacted with the nucleotide of sequencing primer, polymerase and at least one type come pair The template of the template or amplification that are connected to support is sequenced.In some embodiments, sequencing includes by template or amplification Template or extension primer and sequencing primer, polymerase and with it is only one kind of do not include exogenous marker or chain termination base The nucleotide of group is contacted.

Optionally, template (or product of amplification) can be placed in, be confined to or be located at site.In some embodiments, more Kind template/amplification template/extension the first primer is placed in or different sites in site array.In some embodiments In, it placed, positioned or is localized before template amplification.In some embodiments, it is placed after amplification, is fixed Position or localization.For example, the template of amplification or the first primer of extension can be placed in, are located at or be confined to the different loci of array.

Method disclosed herein leads to the generation of multiple amplicons, and at least some amplicons include expanding with cloning Nucleic acid group.The group expanded to the clone generated by the method for present disclosure can be used for a variety of purposes.In some embodiments In, disclosed method (and relevant composition, system and kit) optionally further includes the group's (amplicon) expanded with cloning Analysis and/or processing.For example, in some embodiments, detectable and optionally quantitative display goes out certain desired characteristics The quantity of amplicon.

In some embodiments, method may include measuring which discontinuous support (such as globule) includes amplification Son.Similarly, it includes amplicon that method, which may include which site of measurement array,.Optionally use the detection journey based on DNA Sequence such as UV absorption, with DNA specific dye dyeing,Measurement, qPCR, hybridize with fluorescence probe Carry out the presence of the amplicon on detection assay support or site.In some embodiments, method may include which is small for measurement Pearl support (or site of array) has obtained the amplicon of substantially monoclonal.For example, globule support (or array can be analyzed Site) with determine which support or site can produce detectable and relevant (coherent) (i.e. analyzable) sequence according to Rely property signal.

In some embodiments, the sequencing of the product expanded after amplification.The product for the amplification being sequenced can wrap Include the amplicon comprising the substantially nucleic acid group of monoclonal.In some embodiments, disclosed method includes by multiple amplifications The single member of son forms or is positioned at different sites.Different sites is optionally formed the part of site array.Some In embodiment, the site in site array includes the hole (reaction chamber) on the showing of isFET array.

In some embodiments, the method for downstream analysis includes multiple amplicons are sequenced in parallel at least some.Appoint Selection of land, sequencing is located at multiple template/amplification template/extension the first primer of different array sites in parallel.

In some embodiments, sequencing may include that sequencing primer is bound to at least two different amplicons or at least The nucleic acid of the group of two different substantially monoclonals.

In some embodiments, sequencing may include that nucleotide is incorporated to sequencing primer using polymerase.Optionally, it is incorporated to By-product is incorporated to including forming at least one nucleotide.

Optionally, nucleic acid to be sequenced is located at site.Site may include reaction chamber or hole.Site can be similar or phase With the part of the array in site.Array may include in surface (such as flow cell, electronic device, transistor chip, reaction chamber, slot Deng surface) on site two-dimensional array or matrix or other mediums (such as solid, semisolid, liquid, fluid etc.) in Site cubical array.

In some embodiments, site is operationally coupled to sensor.Method may include detecting core using sensor Thuja acid is incorporated to.Optionally, site and sensor are located in the array in the site for being coupled to sensor.

In some embodiments, site is hydrophilic within the hole for being operationally coupled to sensor comprising being conformally placed in Polymer substrate.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, hydrophilic polymer matrix is in-situ solidifying polymer substrate.

Optionally, hydrophilic polymer matrix includes polyacrylamide, its copolymer, its derivative or combinations thereof.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensor includes field effect transistor (FET).FET may include ion-sensitive FET (ISFET)。

In some embodiments, method (and relevant composition, system and kit) may include optionally using FET It detects one or more nucleotide and is incorporated to presence of the by-product at array site.

In some embodiments, method may include optionally being occurred at least one reaction chamber using FET detection PH variation.

In some embodiments, disclosed method includes that nucleotide is introduced site；With detection because of nucleotide to sequencing Output signal from sensor caused by being incorporated to of primer.Output signal is optionally based on the threshold voltage of FET.Some In embodiment, FET includes the floating gate conductor for being coupled to site.

In some embodiments, FET includes comprising electrical coupling each other and by the floating of multiple conductors of dielectric layer separation Moving grid structure, and floating gate conductor is uppermost conductor in multiple conductors.

In some embodiments, floating gate conductor includes the upper surface for defining the bottom surface in site.

In some embodiments, floating gate conductor includes conductive material, and the upper surface of floating gate conductor includes to lead The oxide of electric material.

In some embodiments, floating gate conductor is coupled at least one reaction chamber by sensing material.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is sensitive to hydrogen ion.

In some embodiments, amplification reaction mixture may include recombinase.Recombinase may include it is any it is appropriate can Promote the reagent of the recombination between polynucleotide molecule.Recombinase can be the enzyme of catalysis homologous recombination.For example, amplified reaction is mixed Close object may include recombinase, the recombinase include or be derived from bacterium, eucaryote or virus (such as bacteriophage) recombination Enzyme.

In some embodiments, amplification reaction mixture includes compound to be formed in combination with primer and polynucleotide template Object can be catalyzed the chain intrusion of polynucleotide template to form the enzyme of D- ring structure.In some embodiments, amplified reaction is mixed Closing object includes one or more albumen for being selected from UvsX, RecA and Rad51.

In some embodiments, amplification reaction mixture may include recombinase auxilin such as UvsY.

In some embodiments, amplification reaction mixture may include single strand binding protein (SSBP).

In some embodiments, amplification reaction mixture may include polymerase.Polymerase optionally has or lacks outer Cut nuclease.In some embodiments, polymerase has 5 ' to 3 ' exonuclease activities, 3 ' to 5 ' exonucleases Activity has above two activity.Optionally, it polymerize any one or more of exonuclease activity as azymia.

In some embodiments, polymerase has strand-displacement activity.

In some embodiments, amplification reaction mixture may include one or more solid or semisolid supports.Branch Hold the first primer of at least one to may include one or more include the first primer sequence of object.In some embodiments, instead Answering at least one polynucleotides in mixture includes the first primer binding sequence.The first primer binding sequence can be with the first primer Sequence is substantially the same or is substantially complementary.In some embodiments, support at least one, it is some or all of Support includes multiple the first primers substantially identical to one another.In some embodiments, all primers on support that This is that substantially the same or all primers include substantially the same the first primer sequence.

In some embodiments, at least one support includes two or more different primers for being connected to it. For example, at least one support may include at least one the first primer and at least one second primer.

In some embodiments, the water phase of reaction mixture includes multiple supports, and plurality of support is at least Two supports are connected to the primer comprising the first primer sequence.In some embodiments, reaction mixture include two or More different polynucleotide templates with the first primer binding sequence.

In some embodiments, amplification reaction mixture may include diffusion limitation agent.Diffusion limitation agent can be effectively Ground prevents or slows down the one or more of polynucleotide template and/or the one or more of amplified reaction product passes through amplified reaction Any reagent of mixture diffusion.

In some embodiments, amplification reaction mixture may include screening agent.Screening agent can be effectively screening and deposit It is one of amplification reaction mixture or a variety of polynucleotides (such as amplified reaction product and/or polynucleotide template) Any reagent.In some embodiments, it sieves agent limitation or slows down polynucleotide amplification product and pass through reaction mixture migration.

In some embodiments, amplification reaction mixture may include crowding agent.

In some embodiments, amplification reaction mixture includes crowding agent and screening agent.

In some embodiments, disclosed method includes expanding two or more multicores to clone by the following method At least two of thuja acid template: (a) formed include single Continuous Liquid Phase amplification reaction mixture, the liquid phase include two or More polynucleotide templates, one or more surfaces or support and amplification component；(b) on one or more supports At least two polynucleotide templates of clone ground amplification.Optionally, the amplification of clone ground includes forming at least two different bases The amplicon of monoclonal in sheet.In some embodiments, the amplification of clone ground includes making amplification reaction mixture experience amplification item Part.In some embodiments, two or more described amplicons are each attached to surface or support.For example, amplification is anti- Answering mixture may include single support or surface, so that each polynucleotide template is connected to the given of support or surface Region.

In some embodiments, the method for nucleic acid amplification can be carried out in single reaction vessel.

In some embodiments, the method for nucleic acid amplification, the single company can be carried out in single Continuous Liquid Phase Continuous liquid phase does not provide the division of the multiple nucleic acid amplification reactions occurred in single reaction vessel.In some embodiments, may be used The method for nucleic acid amplification is carried out in the water-in-oil emulsion (micro- reaction vessel) divided is provided.

In some embodiments, the method for nucleic acid amplification can be carried out so that multiple polynucleotides are connected to support Or surface.For example, method may include forming the reaction mixture comprising at least one surface, and make reaction mixture experience amplification Condition.In some embodiments, surface includes the plane surface or inner wall of the surface of globule, slot or pipe.

It in some embodiments, include: that (a) forms the amplification comprising single Continuous Liquid Phase for the method for nucleic acid amplification Reaction mixture, the liquid phase include multiple globules, multiple and different polynucleotides and recombinase；(b) make amplified reaction mixed It closes object and undergoes isothermal duplication condition, to generate multiple globules for being connected to and being attached to its nucleic acid group of substantially monoclonal.

In some embodiments, present disclosure generally relates to directly carry out on the surface of array nucleic acid-templated The amplification based on array, include the amplicon (amplified production comprising substantially monoclonal so as to cause its any personal feature Group) array formation method (and relevant composition, system and kit).These embodiments with it is described herein Other embodiments form comparison, nucleic acid-templated optionally in the solution in discontinuous in other embodiments It is amplified on support (such as globule), is then assigned into array.

In some embodiments, provide for the amplification based on array method (and relevant composition, system and Kit).In some embodiments, different polynucleotide templates is distributed into site array and then carries out expansion in situ Increase.Then generated amplification subarray is analyzed using downstream program appropriate.

It in some embodiments, include: a) by introducing single polynucleotides each other for the method for nucleic acid amplification At least two sites being in fluid communication distribute at least two different polynucleotides to site；(b) by described in amplification Polynucleotides at least two sites form the nucleic acid groups of at least two substantially monoclonals.Site optionally includes table Face, hole, ditch, flow cell, reaction chamber or slot.In some embodiments, expand site in closed situation each other Increase.For example, at least two sites can keep each other being in fluid communication during amplification.

It in some embodiments, include: a) by introducing single polynucleotides at least for the method for nucleic acid amplification Two sites distribute at least two different polynucleotides to site；(b) by expanding at least two site Polynucleotides form the nucleic acid groups of at least two substantially monoclonals.Site optionally includes surface, hole, ditch, flowing Pond, reaction chamber or slot.In some embodiments, expand site in closed situation each other.For example, at least two A site can keep each other being in fluid communication during amplification.

In some embodiments, site includes reaction chamber, and the method for nucleic acid amplification includes: a) by will be single A polynucleotides introduce at least two reaction chambers being in fluid communication with each other and distribute at least two polynucleotide templates to reaction Room；(b) at least two substantially monoclonals are formed by expanding at least two indoor polynucleotide template of reaction Nucleic acid group.In some embodiments, expand reaction chamber in closed situation each other.For example, at least two Reaction chamber can keep each other being in fluid communication during amplification.

In some embodiments, site includes reaction chamber, and the method for nucleic acid amplification includes: a) by will be single A polynucleotides introduce at least two reaction chambers being in fluid communication with each other and distribute at least two different polynucleotides to anti- Answer room；(b) at least two substantially monoclonals are formed by expanding at least two indoor polynucleotides of reaction Nucleic acid group.In some embodiments, expand reaction chamber in closed situation each other.For example, at least two is anti- Answer room that can keep each other being in fluid communication during amplification.

In some embodiments, any and methodical amplification step of present disclosure can be endless during amplification It is carried out in the case where full denatured polynucleotide.For example, disclosed method may include expanding at least two by isothermal duplication not Same polynucleotides.Amplification may include at least two different polynucleotides of amplification under conditions of substantially isothermal.Optionally, It is expanded in the case where not contacting polynucleotides with chemical denaturant during amplification.

In some embodiments, amplification, which is optionally included in site or reaction chamber, carries out two different amplifications time It closes.For example, amplification may include carrying out at least one RPA bout simultaneously in site or reaction chamber with any sequence of bout or combination At least one template walking bout is carried out in site or reaction chamber.In some embodiments, any one or more are expanded back At least two continuous circulate under substantially isothermy in conjunction carry out.In some embodiments, bout is expanded at least One carries out under substantially isothermy.

In some embodiments, amplification includes contacting polynucleotides to be amplified with reaction mixture.Contact can Optionally carry out before a distribution or later；It will be understood that present disclosure includes wherein by each of polynucleotides and reaction mixture The embodiment that a component (or combination of component) is continuously contacted in different times, and wherein by reaction mixture Either one or two of or some components polynucleotides different at least two contact before dispensing and by remaining group of reaction mixture The embodiment for dividing the polynucleotides different at least two to contact after dispensing.

At least two assigned polynucleotides are used as nucleic acid synthesis optionally in its respective reaction chamber Template.In some embodiments, at least two polynucleotides include different sequences.In some embodiments, multicore glycosides Acid is double-strand or as single-stranded before a distribution.In some embodiments, polynucleotides are linear, cricoid or both Combination.In a typical implementation, polynucleotides be at least partly double-strand (or with single stranded form distribute and then dividing With later making its at least partly double-strand in site or reaction chamber).It can make polynucleotides double-strand before amplification (especially Ground is in the embodiment that wherein amplification includes RPA or template walking).

In some embodiments, at least two different polynucleotide templates to be amplified respectively contain primer combination Position, and expanding includes that primer is bound to primer binding site to form primer-template double-strand.

Optionally, amplification includes extension primer-template double-strand primer.Extend and optionally occurs in the site of array or anti- Answer at room or within.Optionally, extension primer includes by primer and polymerase and the nucleotide of one or more types in nucleosides Acid is contacted under the conditions of being incorporated to.In some embodiments, the nucleotide of one or more types does not include exogenous marker, special Not optically detectable label, such as fluorescence part or dyestuff.Optionally, reaction mixture includes nucleotide, is day So existing nucleotide.Optionally, nucleotide does not include group (such as the double deoxidation group, reversible termination for terminating nucleic acid synthesis Son etc.).Normally, extension primer is occurred with template dependent manner.

Optionally, at least two different polynucleotides (template i.e. to be amplified) are respectively contained with the first primer extremely Certain few First ray (being referred to as " the first primer binding site ") that is a part of substantially the same or being substantially complementary.

In some embodiments, reaction mixture includes the first primer comprising the first primer sequence.The first primer is appointed Selection of land includes extendible end (such as 3 ' ends containing OH).Optionally by the first primer be connected to compound (such as " resistance Power label ") or be connected on support (such as globule or site or surface of reaction chamber).

In some embodiments, at least two different polynucleotides include at least certain a part of base with the second primer Identical or the second sequence (be referred to as " the second primer binding site ") for being substantially complementary in sheet.

In some embodiments, method includes that one or more nucleotide are incorporated to second by using polymerase to draw The second primer of object-template double-strand extends the second primer, to form the second primer of extension.

In some embodiments, do not make it is nucleic acid-templated at least two, three, four or continuous more than four Method is carried out in the case where undergoing extreme Denaturing during nucleic acid synthesis circulation.For example, method may include two, three, four A or continuous nucleic acid synthesis step more than four nucleic acid-templated is contacted without making with chemical denaturant.In some embodiments In, method may include progress two, three, four or the continuous nucleic acid synthesis circulation more than four without making nucleic acid-templated warp Go through the reality higher than template or template group or the Tm being computed (template or template group reality or the average Tm that is computed) it Lower 25,20,15,10,5,2 or 1 DEG C of temperature.Two, three, four or more than four continuous nucleic acid synthesis circulation can wrap Include the partial denaturation and/or primer extension procedures of intervention therebetween.

In some embodiments, disclosed method (and relevant composition, system and kit) may also include one Second primer strand of a or multiple extensions is connected to support.In some embodiments, support is connected to the first primer.Example Such as, support may include multiple the first primers, and method may include the second primer and support for extending at least one The first primer hybridization, so that the second primer of extension is connected to support.For example, the first primer can be hybridized to the second of extension The first primer binding site in primer.Support may include the surface of for example any array.

In some embodiments, disclosed method includes optionally in the identical continuous phase of reaction mixture and in table At the different loci in face (such as in array), by amplification the first template the first amplicon of formation on the first support, and The second amplicon is formed by the second template of amplification on second support.First amplicon optionally connects or is attached to first Object is held, and the second amplicon optionally connects or be attached to the second support.

Two or more different sites that disclosed method is optionally included in site array by cloning expand Two or more polynucleotide templates generate the amplicon of two or more monoclonals or substantially monoclonal, so as to will At least two sites are formed as respectively containing the nucleic acid group of substantially monoclonal.Two or more polynucleotide templates are optionally Be placed in or positioned at different sites and then with the Continuous Liquid Phase of the amplification reaction mixture of array contact in expand with being cloned Increase.The Continuous Liquid Phase of amplification reaction mixture may include continuous aqueous phase.

In some embodiments, amplification includes the polynucleotides group of the substantially amplification of monoclonal of generation at least two, Each of the polynucleotides group is formed by the amplification of single polynucleotide template.

Optionally, the amplification of clone ground includes at least one RPA bout.

Optionally, the amplification of clone ground includes at least one template walking bout.

In some embodiments, multiple and different polynucleotide templates expand it is preposition in or positioned at different site. For example, the template of amplification or the first primer of extension can be placed in, are located at or be confined to the different loci of array.

In some embodiments, amplification leads to be formed at least two in at least two different sites on surface substantially The nucleic acid group (such as amplicon) of upper monoclonal, may then use that nucleic acid group described in program in-situ study appropriate.

In some embodiments, present disclosure generally relate to preparation surface method (and relevant composition, System and kit).Optionally, surface includes multiple sites comprising the first site and the second site.

In some embodiments, method includes forming nucleic acid array on the surface, wherein described formed includes by first Nucleic acid is connected to the first site and the second nucleic acid is connected to the second site.The connection is optionally by using public affairs herein Any method opened, including being for example covalently attached to the primer on surface by the way that nucleic acid to be connected to and carrying out.

In some embodiments, method includes the reagent for synthesizing at least the first and second nucleic acid with comprising being used for nucleic acid Single reaction mixture contact.Reaction mixture is optionally including any one or more components described herein.One In a little embodiments, reaction mixture includes all components needed for carrying out RPA.In some embodiments, reaction mixture All components comprising carrying out template walking.

In some embodiments, method includes by using the reagent duplication for nucleic acid synthesis in reaction mixture First or second nucleic acid to form the first amplicon in the first site and forms the second amplicon in second point.Duplication may include Primer extend.Duplication may include one or more RPA circulations.Duplication may include one or more template walking circulations.

In some embodiments, duplication includes that at least one RPA is recycled.

In some embodiments, duplication includes at least one template walking circulation.

In some embodiments, duplication includes at least one RPA circulation and at least one template walking circulation.

In some embodiments, duplication includes at least one RPA bout.

In some embodiments, duplication includes at least one template walking bout.

In some embodiments, duplication includes at least one RPA bout and at least one template walking bout.

In some embodiments, present disclosure generally relate to preparation surface method (and relevant composition, System and kit), which comprises (a) provides the surface with multiple sites comprising the first site and the second site； (b) nucleic acid array is formed on the surface, wherein the formation includes the first nucleic acid being connected to the first site and by the second nucleic acid It is connected to the second site；(c)；(d) by using in reaction mixture for the reagent duplication first of nucleic acid synthesis or the Two nucleic acid to form the first amplicon in the first site and form the second amplicon in second point.

In some embodiments, the method that present disclosure generally relates to preparation surface, which comprises mention For the surface with multiple sites comprising the first site and the second site；Nucleic acid array is formed on the surface, wherein the shape At including that the first nucleic acid is connected to the first site and the second nucleic acid is connected to the second site；By at least the first and second nucleic acid It is contacted with the single reaction mixture of the reagent comprising being synthesized for nucleic acid；Nucleic acid is used for by using in reaction mixture The reagent amplification first or second nucleic acid of synthesis comes in the substantially amplicon of monoclonal of the first site formation first and second The amplicon of the substantially monoclonal of site formation second.Optionally, the first and second sites keep being in fluid communication during amplification. Optionally, it is expanded in the case where incomplete denatured polynucleotide during amplification.For example, disclosed method may include passing through Isothermal duplication expands at least two different polynucleotides.Amplification may include that at least two are expanded under conditions of substantially isothermal A different polynucleotides.Optionally, expand in the case where contact polynucleotides with chemical denaturant during amplification Increase.

In some embodiments, at least one site in multiple sites includes reacting hole, trough or room.

In some embodiments, at least one site in multiple sites is connected to sensor.

In some embodiments, sensor is able to detect the nucleotide occurred at or near at least one site simultaneously Enter.

In some embodiments, sensor includes field effect transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites include to be connected to surface Primer.

In some embodiments, at least one site in multiple sites includes and is conformally placed in operationally to be coupled to biography Hydrophilic polymer matrix within the hole of sensor.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensor includes field effect transistor (FET).FET may include ion-sensitive FET (ISFET), chemFET, bioFET etc..

In some embodiments, FET is able to detect nucleotide and is incorporated to by-product in the presence at least one site.

In some embodiments, FET is able to detect the chemical part selected from hydrogen ion, pyrophosphate, hydroxidion etc..

In some embodiments, method may include optionally using FET detection at site or at least one reaction The pH of indoor generation changes.

In some embodiments, disclosed method includes that nucleotide is introduced at least one site in multiple sites；With Detect the output signal from sensor because of caused by being incorporated to of nucleotide to sequencing primer.Output signal is optionally based on The threshold voltage of FET.In some embodiments, FET includes the floating gate conductor for being coupled to site.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is sensitive to hydrogen ion.

In some embodiments, reaction mixture includes all components needed for carrying out RPA.

In some embodiments, reaction mixture includes all components needed for carrying out template walking.

In some embodiments, reaction mixture may include one or more solid or semisolid supports.Support At least one to may include one or more include the first primer sequence the first primer.In some embodiments, support At least one include two or more different primers for being connected to it.For example, at least one support may include at least One the first primer and at least one second primer.

Alternatively, in some embodiments, reaction mixture does not include any support.In some embodiments, directly Connect at least two different polynucleotide templates of amplification on the site of array or the surface of reaction chamber.

In some embodiments, reaction mixture may include recombinase.Recombinase may include any appropriate can promote The reagent of recombination between polynucleotide molecule.Recombinase can be the enzyme of catalysis homologous recombination.For example, reaction mixture can wrap Containing recombinase, the recombinase include or be derived from bacterium, eucaryote or virus (such as bacteriophage) recombinase.

Optionally, reaction mixture includes the nucleotide for not carrying out exogenous marker.For example, nucleotide can be it is naturally occurring Nucleotide, or synthetic analogues of the optically detectable label not comprising fluorescence part, dyestuff or other external sources.Optionally Ground, reaction mixture include nucleotide, are naturally occurring nucleotide.Optionally, nucleotide, which does not include, terminates nucleic acid synthesis Group (such as double deoxidation group, reversible terminator etc.).

Optionally, reaction mixture includes nucleotide, is naturally occurring nucleotide.Optionally, nucleotide does not include Terminate the group (such as double deoxidation group, reversible terminator etc.) of nucleic acid synthesis.

In some embodiments, reaction mixture include in combination with primer and polynucleotide template with formed compound or The chain intrusion of polynucleotide template can be catalyzed to form the enzyme of D- ring structure.In some embodiments, reaction mixture includes One or more is selected from the albumen of UvsX, RecA and Rad51.

In some embodiments, present disclosure generally relate to preparation surface method (and relevant composition, System and kit), which comprises (a) provides the surface with multiple sites, draws wherein each site is connected to nucleic acid Object；(c) surface is contacted with multiple polynucleotide templates and at least one template is connected to surface；It is expanded on the surface A few template, to form the target polynucleotide of the amplification of the substantially monoclonal at the site that at least one is located on surface The group of sequence.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, comprising: (1) tool is provided There is the surface in the first site and the second site, the first site is operationally coupled to first sensor and includes the first template；The Two sites are operationally coupled to second sensor and include the second template；(2) reaction mixture is distributed to first and second Site；(3) the first amplicon is formed by expanding the first template in the first site；With by the second site expand the second mould Plate forms the second amplicon.

In some embodiments, amplification includes that at least one RPA is recycled.

In some embodiments, amplification includes at least one template walking circulation.

In some embodiments, amplification includes at least one RPA circulation and at least one template walking circulation.

In some embodiments, amplification includes at least one RPA bout.

In some embodiments, amplification includes at least one template walking bout.

In some embodiments, amplification includes at least one RPA bout and at least one template walking bout.

In some embodiments, disclosed herein some or all of methods can lead to the generation of multiple amplicons, At least some nucleic acid groups comprising the amplification of clone ground of the amplicon.Expand to the clone generated by the method for present disclosure The group of increasing can be used for a variety of purposes.In some embodiments, disclosed method (and relevant composition, system and kit) It optionally further include the analysis and/or processing of the group's (amplicon) expanded with cloning.

In some embodiments, the amplicon generated according to present disclosure experience downstream analysis method can be made for example to survey Sequence.

In some embodiments, can at the site of distribution further the nucleic acid of analysis (such as sequencing) amplification without The product of amplification is recycled and moves to different site or surface to be used to analyze (such as sequencing).

In some embodiments, the method for downstream analysis includes that at least part of multiple amplicons is sequenced in parallel. Optionally, the multiple template/amplification template/extension the first primer for being located at different array sites is sequenced in parallel.

For example, in some embodiments, the product in situ that amplification is sequenced after amplification.The product for the amplification being sequenced can Amplicon including the nucleic acid group comprising substantially monoclonal.Optionally, the list for being located at the different loci of array is sequenced in parallel The nucleic acid group (amplicon) of clone.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is sensitive to hydrogen ion.

In some embodiments, reaction mixture may include recombinase auxilin such as UvsY.

In some embodiments, reaction mixture may include single strand binding protein (SSBP).

In some embodiments, reaction mixture may include polymerase.Polymerase optionally has or lacks circumscribed core Phytase activity.In some embodiments, polymerase has 5 ' to 3 ' exonuclease activities, 3 ' to 5 ' exonuclease activities Or there is above two activity.Optionally, it polymerize any one or more of exonuclease activity as azymia.

In some embodiments, polymerase has strand-displacement activity.

In some embodiments, reaction mixture may include diffusion limitation agent.Diffusion limitation agent can be effective ground resistance Only or slow down polynucleotide template one or more and/or amplified reaction product one or more pass through reaction mixture expand Scattered any reagent.

In some embodiments, reaction mixture may include screening agent.Screening agent can be effectively screening and be present in Any examination of one of reaction mixture or a variety of polynucleotides (such as amplified reaction product and/or polynucleotide template) Agent.In some embodiments, it sieves agent limitation or slows down polynucleotide amplification product and pass through reaction mixture migration.

In some embodiments, reaction mixture may include crowding agent.

In some embodiments, reaction mixture includes crowding agent and screening agent.

In some embodiments, disclosed method includes by each and recombinase of at least two polynucleotides, even Being connected to the supports of multiple first Oligonucleolide primers, (the first Oligonucleolide primers are at least partially complementary to polynucleotides at least Certain a part), polymerase and multiple nucleotide contacted in any order and with any combination.

In some embodiments, at least two different polynucleotides include the forward direction comprising the first primer binding site Chain, and the amplification at least two sites (or at least two reaction chambers) is optionally included in site or reaction chamber The first primer is formed into the first primer-template double-strand in conjunction with the first primer binding site.Optionally, the first primer is at least The combination of two different polynucleotide templates is by recombinase-mediated.For example, amplification may include being formed comprising recombinase and first The nucleoprotein complex of primer.Optionally, the first primer is connected to the surface of site or reaction chamber.In some embodiments, It include: to form the first nucleoprotein complex (or " first nucleoprotein filament ") in site or the indoor amplification of reaction.Amplification is optionally Further include by the polynucleotides in site or reaction chamber at least one with the first nucleoprotein filament, polymerase and multiple nucleotide with Any sequence or combination are contacted.

Optionally, the support (such as globule) that the dispersion of multiple the first primers will be respectively contained before amplification respectively divides It is assigned in reaction chamber or site, and expanding includes different more of amplification at least two on site or the indoor support of reaction One of nucleotide.In some embodiments, can before amplification either one or two of duplicate allocation and/or contact procedure, optionally Ground generates site or the quantity and/or yield of reaction chamber of monoclonal product to increase.

Optionally, amplification includes extending the first primer-template double-strand the first primer using polymerase in reaction chamber, from And form the first primer extended.Optionally, extend the first primer and replace reverse strand from positive chain.The first primer of extension is optional Ground includes the second primer binding site.

Optionally, amplification includes inverse composition step, and the step includes that the second primer is bound to the first of extension to draw Second primer binding site of object, and extend the second primer to form the second primer-template double-strand.Optionally, the second primer is extremely The combination of polynucleotide template is by recombinase-mediated.For example, amplification may include forming the core egg comprising recombinase and the second primer White compound.Optionally, the second primer is connected to the surface of site or reaction chamber.In some embodiments, in site or instead Answering indoor amplification includes: to form the second nucleoprotein complex (or " second nucleoprotein filament ").Amplification optionally further includes by position At least one of at least one or the first primer that extends of point or polynucleotide template in reaction chamber and the second nucleoprotein filament, Polymerase and multiple nucleotide are in any order or combination is contacted.

Optionally, amplification includes extending the first primer-template double-strand, the second primer-template double-strand using polymerase or prolonging It stretches both above-mentioned.Polymerase can have strand-displacement activity.

In some embodiments, method (and relevant composition, system and kit) may include by least one base The group of monoclonal is placed in, is positioned at or is confined to site in sheet.Site can form the part of site array.

Optionally, at least one of site includes reaction chamber, support, particle, particle, sphere, globule, filter, flowing Pond, hole, ditch, tank therefor (channel reservoir), gel or pipe inner wall.

In some embodiments, at least one site include conformally be placed in operationally be coupled to sensor hole it Interior hydrophilic polymer matrix.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is sensitive to hydrogen ion.

Optionally, multiple and different polynucleotide template (or polynucleotides of amplification) contains optional comprising at least one Property cutting part nucleic acid.

Optionally, the part of alternative cutting includes uracil.

It optionally, further include that cleavable part is cut with cutting agent for the method for nucleic acid amplification.

Optionally, cutting can for example carry out before forming reaction mixture before amplification.

Optionally, cutting can be carried out for example after nucleic acid-templated be amplified after amplification.

Optionally, reaction mixture includes that at least one contains the primer of cleavable part.

Optionally, multiple and different polynucleotides include multiple amplicons.

Optionally, multiple and different polynucleotides include multiple and different amplicons.

In some embodiments, the U.S. Provisional Patent Application No. 61/792247 that on March 15th, 2013 can be used to submit Any method, composition, system and kit disclosed in (being incorporated herein by reference in their entirety) are expanded.

In some embodiments, the amplicon experience downstream analysis method generated according to present disclosure can be made for example fixed Amount.For example, in some embodiments, the quantity of the detectable amplicon for going out certain desired characteristics with optionally quantitative display.

In some embodiments, the nucleic acid of amplification is optionally made to undergo other downstream analysis in the disclosed methods Step.

It is some include the embodiment that different polynucleotide templates is expanded on discontinuous and isolated support In, method may include measuring which discontinuous support (such as globule) includes amplicon.Similarly, template is expanding wherein It is assigned before increasing into the embodiment of array, method may include which site of measurement array includes amplicon, and can be appointed Selection of land further includes counting the quantity in the site comprising amplicon.Optionally use for example ultraviolet suction of the detection program based on DNA Receive, dyed with DNA specific dye,Measurement, qPCR, carry out detection assay branch with hybridizing etc. for fluorescence probe Hold the presence of the amplicon on object or site.In some embodiments, method may include measuring which globule support (or battle array The site of column) obtain the amplicon of substantially monoclonal.For example, globule support (or array site) can be analyzed so which to be determined A little supports or site can produce detectable and relevant (i.e. analyzable) sequence dependent signal.

In some embodiments, disclosed method includes other downstream analysis step, and the downstream analysis step mentions For previously passed routine techniques such as digital pcr or number RPA (as example in Shen 2011Analytical Chemistry 83:3533-3540；(all documents pass through reference to U.S. Patent Application Publication US2012/0264132 and 2012/0329038 It is integrally incorporated herein) described in) information of the same type of acquisition.Digital pcr (dPCR) is that Conventional polymerase chain formula is anti- The improvement for answering (PCR) can be used for direct quantitative and clone's ground amplification of nucleic acid (including DNA, cDNA, methylate DNA or RNA). A difference between dPCR and normal PCR is the method for measuring nucleic acid amount.The every single sample of PCR and dPCR all carries out one Reaction, dPCR is also individually reacted in sample, however sample is separated into a large amount of subregion and in each subregion Respectively reacted.The sensitive measurement for separately allowing nucleic acid amount.Being proved dPCR can be used for studying in gene order Variation, such as copy number variation or point mutation.

Compared with this method, dPCR usually requires subregion of the sample before amplification；On the contrary, several implementations disclosed herein Scheme provides parallel amplification of the different templates in the single continuous phase of reaction mixture without subregion.In dPCR, Sample is usually partitioned for the positioning of the individual nucleic acid molecules in sample and is confined in many isolated regions.By simple Dilution method divides sample so that each part includes about 1 DNA profiling copied or less.By separating individual DNA profiling, This method is effectively enriched in original sample with DNA molecular existing for extremely low level.The subregion of sample be conducive to using The statistical numerator counts of Poisson.As a result, each subregion will respectively include " 0 " or " 1 " molecule, or it is negative or Positive reaction.Although the starting copies quantity of molecule is proportional to the quantity of amplification cycles in Standard PCR, dPCR is usual The sample size of starting is determined independent of the quantity of amplification cycles.

The conventional method of dPCR analysis usually identifies the product of amplification using fluorescence exploration and based on the detection method of light. Such method needs the abundant amplification of target molecule to generate enough signals being detected, but can lead to additional mistake or Deviation.

It include the nucleic acid-templated distribution into the hole of isFET array and subsequent template in battle array in those of present disclosure In the embodiment of amplification in the hole of column, optional downstream analysis step can be carried out after amplification, quantitatively include amplification The site of product or the quantity in hole.In some embodiments, can detect the product of nucleic acid amplification reaction to count includes amplification Template site or hole quantity.

For example, in some embodiments, present disclosure is usually directed to nucleic acid synthetic method, comprising: offer includes The sample of the polynucleotides of first quantity；It distributes with by the single polynucleotides of sample into the different loci of site array.

Optionally, method may also include is formed substantially singly by expanding single polynucleotides in their own site The nucleic acid group of clone.

Optionally, site keeps being in fluid communication during amplification.

Optionally, amplification includes partial denaturation template.

Optionally, amplification includes that template is made to undergo partial denaturation temperature.In some embodiments, template includes comprising drawing The eutectic point sequence of object binding site, when template undergoes partial denaturation temperature, the sequence is endowed single-stranded.

Optionally, amplification includes partial denaturation template.

Optionally, amplification includes by the different template of at least two at two different array sites and being used for nucleic acid The single reaction mixture contact of amplification.

Optionally, reaction mixture includes recombinase.

Optionally, it includes the primer of " resistance label " that reaction mixture, which includes at least one,.

Optionally, amplification includes carrying out at least one amplification cycles, and the amplification cycles include: partial denaturation template, are made Primer hybridizes with template, and with template dependent manner extension primer.Optionally, amplification includes isothermal duplication.In some implementations In scheme, expanded under conditions of substantially isothermal.

In some embodiments, the percentage in the site containing one or more template molecules is greater than 50% and is less than 100%.

In some embodiments, disclosed method, which may also include, detects at least one site because at least one amplification follows The variation of ion concentration caused by ring.

In some embodiments, disclosed method may also include the initial amount of quantitative target nucleic acid.

It is found in such as 2012 4 using some examples of the digital pcr based on array of the sensing technology based on ion In the U.S. Provisional Application No. 61/635584 (being incorporated herein by reference in their entirety) that the moon is submitted on the 19th.

In some embodiments, the method that present disclosure generally relates to detection target nucleic acid, comprising: draw sample Divide into multiple sample sizes, wherein the part more than 50% includes the target nucleic acid molecule that every sample size is not more than 1；Make more Condition of a sample size experience for amplification, wherein the condition includes partial denaturation condition；Wherein there is target nucleic acid in detection Sample size in ion concentration variation；Count the quantity with the part of target nucleic acid of amplification；With target in determining sample The amount of nucleic acid.The variation of ion concentration can be the raising of ion concentration or can be the reduction of ion concentration.In some implementations In scheme, method may also include sample and bead set.In some embodiments, method may include that sample is loaded onto substrate On, wherein the substrate includes at least one hole.

In some embodiments, making target nucleic acid experience partial denaturation condition includes by target nucleic acid molecule their own It is contacted under the conditions of RPA in sample size with recombinase and polymerase.

In some embodiments, making target nucleic acid experience partial denaturation condition includes that target nucleic acid molecule is made to undergo partial denaturation Temperature.

In some embodiments, present disclosure generally relates to the method for carrying out the absolute quantitation of nucleic acid, comprising: Nucleic acid-templated sample of the dilution comprising initial amount and by multiple sites of the nucleic acid-templated distribution of sample to array, wherein wrapping Percentage containing one or more nucleic acid-templated sites is greater than 50% and less than 100%；Multiple sites are made to undergo at least one Amplification cycles, wherein carrying out the amplification cycles according to any amplification method disclosed herein；Detect multiple sample sizes At least one in because of caused by least one amplification cycles ion concentration variation；With the starting number of quantitative nucleic acid template Amount.The variation of ion concentration can be the raising of ion concentration, the reduction of ion concentration, the variation of pH, can be related to positive ions Such as the detection of hydrogen ion, anion such as pyrophosphate molecule or cation and anion.

In some embodiments, present disclosure also generally relates to incite somebody to action for the chain exchange by using recombinase-mediated The different supports or the different positions being connected in multiple sites that the individual member of nucleic acid-templated group is connected in multiple supports The method (and relevant composition, system and kit) of point.These methods, composition, system and kit can be in needs It does not obtain or distinguishes in the application of different amplicons for generating the amplification subgroup of the fixation suitable for operation.In some realities It applies in scheme, multiple sites in multiple discontinuous supports or array respectively contain capture primer.Each template is to each The fixation of support (or to each site of array) can by by template with support or site primer (" merging primer ") In the presence of contacted to realize.In some embodiments, fusion primer includes the target for being complementary to the part of template Specificity portion, and it is complementary to the universal primer binding site of capture primer at least certain a part in support or site.Optionally Ground is contacted in the presence of RPA component.RPA component may include recombinase.RPA component may include strand displacement polymerization Enzyme.In some embodiments, fusion primer is recombinated into template by the chain exchange of recombinase-mediated, so that formation includes The template of universal primer binding site: primer adduct.In some embodiments, then capture primer is recombinated to general and is drawn In object binding site, the template for being connected to the fixation in support or site is formed.

In some embodiments of the amplification based on globule, different target specific moiety and common will be respectively contained The fusion primed libraries of universal primer binding site polymerize with multiple template and multiple supports comprising polymerase and strand displacement It is contacted in the reaction mixture of enzyme.Then by making mixture undergo RPA condition by template library connection to multiple supports Object, to generate the multiple supports for being respectively connected with different templates.

In some embodiments of the amplification based on array, different target specific moiety and common will be respectively contained The fusion primed libraries and multiple template of universal primer binding site and the surface comprising multiple sites are including polymerase and chain It is contacted in the reaction mixture of metathesis polymerizable enzyme.At least some of multiple sites include general capture primer.Then pass through Make mixture experience RPA condition by multiple sites of template library connection to surface, is respectively connected with different moulds to generate Multiple supports of plate.

In some embodiments, present disclosure is usually directed to comprising for being expanded in parallel using partial denaturation condition One or more nucleic acid-templated reagents composition (and it is relevant be used to prepare and using the composition method).

In some embodiments, composition may include described herein for carrying out any component of the component of RPA.

In some embodiments, composition may include described herein for carrying out any of the component of template walking Component.

In some embodiments, present disclosure generally relates to the composition and system of nucleic acid amplification, comprising: packet Surface containing the first site and the second site；And nucleic acid amplification reaction mixture, wherein the mixture and first and second Point contact.

In some embodiments, reaction mixture includes recombinase.

In some embodiments, the first site is operationally coupled to first sensor, and the second site can operate Ground is connected to second sensor.

In some embodiments, the first and second sites are operably coupled to identical sensor.

Optionally, the first site includes the nucleic acid group of the first substantially monoclonal.Second site optionally includes the second base The nucleic acid group of monoclonal in sheet.

In some embodiments, disclosed composition includes: the surface comprising the first site and the second site, wherein the One site include first substantially the nucleic acid group of monoclonal and the second site include the second substantially monoclonal nucleic acid group；With Nucleic acid amplification reaction mixture, wherein the mixture is contacted with the first and second sites.

In some embodiments, composition includes the array in site, and the array in the site includes comprising (such as connecting Extremely) the first site of the first capture primer and the second site comprising the capture primer of (such as being connected to) second.

In some embodiments, sensor includes field effect transistor (FET).

In some embodiments, at least the first site or the second site or the first and second sites include to be connected to surface Capture primer.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is sensitive to hydrogen ion.

In some embodiments, the method for amplification may include carrying out the public affairs of the United States Patent (USP) as disclosed on June 21st, 2012 " template walking " described in the number of opening 2012/0156728 (it is incorporated herein by reference in their entirety).For example, in some implementations In scheme, present disclosure expands one or more nucleic acid-templated cores to expand with forming clone with generally relating to clone Method, composition, system, device and the kit of acid template group.Any amplification method described herein optionally includes weight Multiple nucleic acid amplification circulation.Amplification cycles optionally include: (a) hybridization of primer to template strand, and (b) primer extend is to form One chain extended, (c) partially or incompletely denaturation of the chain extended from template strand.The primer of template strand is hybridized to (for convenience of rising See, be named as " forward direction " primer) optionally fix on the support or be fixed to support.Support is, for example, that solid or half are solid Body.Optionally, from step (c) template strand denaturation portion in next amplification cycles can freely from different forward directions Primer hybridization.In embodiments, in subsequent amplification cycles primer extend include the first extension chain setting from template strand It changes.It may include such as second " reversed " primer, be hybridized to 3 ' ends of the chain of the first extension.Reverse primer is optionally non-solid Fixed.

In embodiments, using being fixed on one or more solid or semisolid supports or be fixed to its primer Expand template.Optionally primer of the support comprising the fixed first part for being complementary to template strand.Optionally, support is not shown It lands comprising the fixed primer with the second non-overlap homeologous of identical template strand.If two parts do not include any Hybridize or be complementary the subdivision of object hybridization each other, then they are non-overlaps.In another example, support is optionally Indistinctively comprising the fixed primer that can hybridize with the complement of template strand.

Optionally, in single Continuous Liquid Phase in the presence of one or more supports, wherein each is supported Object includes one or more fixed sites, is simultaneously expanded multiple nucleic acid-templated.In embodiments, expand each template with Generate the amplification subgroup of clone, wherein each clone group be fixed on the support different from the group of other amplifications or fixed site it It is interior or on.Optionally, the group of amplification keeps substantially monoclonal after amplification.

Template be for example amplified with generate clone group, it includes the homologous chain of template (referred to herein as " template strand " or " reverse strand ") and/or template complementary chain (referred to herein as " primer strand " or " positive chain ").In embodiments, in institute Keep Clonal by maintaining the association between template strand and its primer strand in the nucleic acid group of the amplification of generation, thus effectively By relevant clone generation joint or " constraint " together and a possibility that reduce cross contamination between different clone crowd.Optionally Ground, the nucleic acid for cloning one or more amplifications in group are connected to support.The clone group of substantially the same nucleic acid can be optional Ground has macro manifestations spatially limit to or discontinuous.In embodiments, clone group can be similar to isolated point or collection Fall (for example, when being assigned into support, optionally on the outer surface of support).

In some embodiments, present disclosure is usually directed to the novel of the clone group for the limitation for generating clonal expansion Method, the clone group are optionally fixed to one or more supports or are fixed in which or on which.Support can be such as It is solid or semisolid (such as gel or hydrogel).The clone group of amplification be optionally connected to support outer surface or can also In the inner surface of support (such as when support has porous or matrix structure).

In some embodiments, pass through the primer extend along purpose template strand (also referred to as " reversed " chain) of multiple circulations To realize amplification.For convenience, will be hybridized to purpose template strand primer be referred to as " forward direction " primer, and optionally with template according to Property mode is relied to extend it, to form " forward direction " chain for being complementary to purpose template strand.In certain methods, positive chain itself with claimed Make second primer hybridization of " reversed " primer, extends second primer to form new template strand (also referred to as reverse strand).Appoint Selection of land, new template chain is at least partly homologous with original purpose template (" reversed ") chain.

As mentioned, one or more primers can be fixed to one or more supports or be fixed in which or on which. Optionally, a primer is fixed by being connected to support.Second primer can be it is existing and be optionally not fixed or It is connected to support.It can simultaneously be expanded on different supports or fixed site for example in single Continuous Liquid Phase different Template to form the nucleic acid group of monoclonal.If any part of liquid phase and any other part of liquid be fluid contact or Connection, then it is believed that liquid phase is continuous.In another example, if part is not complete with the rest part of liquid Ground subdivision divides or in other ways by fully physically separate, then it is believed that liquid phase is continuous.Optionally, liquid phase be can Flowing.Optionally, Continuous Liquid Phase is not in gel or Medium Culture.In other embodiments, Continuous Liquid Phase is in gel or matrix It is interior.Such as Continuous Liquid Phase occupies the hole, gap or other gaps of solid or semisolid support.

When liquid phase is in gel or Medium Culture, one or more primers are optionally fixed on the support.Optionally support Object is gel or matrix itself.Alternatively, support is not gel or matrix itself.In instances, a primer, which is fixed on, includes On solid support in gel and it is unsecured to gel molecular.Support be, for example, plane surface form or one or Multiple particles.Optionally plane surface or multiple particles include the forward primer for having basically the same sequence.In embodiment party In case, support does not include the second different primer of significant quantity.Optionally, solution of the second on-fixed primer in gel In.Second on-fixed primer is for example bound to template strand (i.e. reverse strand), and fixed primer is bound to positive chain.

The embodiment of template walking includes the method for primer extend, comprising: (a) primer-hybridization step (b) extends step Suddenly, and (c) walk step.Optionally, primer-hybridization step includes being hybridized to the first primer molecule (" the first forward primer ") Complementary forward primer binding sequence (" positive PBS ") in nucleic acid chains (" reverse strand ").Optionally, extending step includes generating The positive chain of first extended, the chain are the overall length complements of reverse strand and are hybridized to it.Such as by using reverse strand as mould Plate extends the first forward primer molecule with template dependent manner to generate the first of extension the positive chain.Optionally, walking step Including the second primer (" the second forward primer ") is hybridized to positive PBS, wherein reverse strand is also hybridized to the first positive chain.Example Such as, walking step includes at least partly (" free fraction ") that forward direction PBS is denaturalized from positive chain, and another part of reverse strand is still So it is hybridized to positive chain.

In embodiments, primer extension method is the amplification method for including template walking, wherein primer-hybridization, extension And/or any one or more steps of walking are repeated at least once.For example, method may include being followed by one or more amplification Circle amplification forward direction chain.Amplification cycles optionally include extension and walking.Exemplary amplification circulation include or substantially by extend and Subsequent walking composition.Optionally, the second forward primer of the first amplification cycles is used as the first forward direction of subsequent amplification cycles Primer.For example, the second forward primer of step of walking in the first amplification cycles is used as the extension step of subsequent amplification cycles First forward primer.

Optionally, primer extend or the method for amplification further include extending as follows or expanding reverse strand: (a) by the The one reverse primer molecule reverse primer binding sequence complementary on the positive chain of extension (" reversed PBS ") hybridization；(b) pass through Use positive chain as template the first reverse primer molecule extended with template dependent manner to generate the first reverse strand of extension, The chain is the overall length complement of positive chain and is hybridized to it；(c) the second primer (" the second reverse primer ") is hybridized to reversely PBS, wherein positive chain is also hybridized to the first reverse strand.The one or many repetitions for optionally carrying out step (b)-(c), wherein walking Suddenly second reverse primer of (c) is first reverse primer of (b) the step of repetition；Wherein in one or many repetition phases Between or between the major part of institute's having time forward direction chain be hybridized to reverse strand.In embodiments, it is largely optionally at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

Optionally, reverse strand and/or positive chain are not exposed to complete Denaturing, the complete denaturation item during amplification Part will lead to multiple chains of signal portion (such as more than 10%, 20%, 30%, 40% or 50%) with its extend and/or it is complete Long complement is kept completely separate.

In embodiments, in one or more amplification cycles, (such as 1,5,10,20 or all amplification of progress is followed Ring) during or between institute having time is positive and/or the major part of reverse strand be optionally hybridized to it is extension and/or overall length mutual Mend object.In embodiments, the major part of chain be optionally chain at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.In embodiments, this T for being higher than the primer not extended by maintaining amplified reaction_mAnd it is lower than Primer-complementary strand T_mTemperature realize.For example, amplification condition is maintained in such temperature, higher than what is do not extended The T of forward primer_mAnd lower than the T of reverse strand extend or overall length_m.Similarly, for example, amplification condition be maintained at it is such In temperature, it is higher than the T for the reverse primer not extended_mAnd lower than the T of positive chain extend or overall length_m。

Optionally, one or more forward primers and/or one or more reverse primers are respirable (breathable), such as with low T_m.In instances, can breathe the nucleotide base of primer at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% is adenine, thymidine or uracil or is complementary to adenine, chest Gland pyrimidine or uracil.

In some embodiments, present disclosure generally relates to clone on the support in amplified reaction solution Method, composition, system, device and the kit of ground amplification of nucleic acid template.Optionally, nucleic acid-templated and support is comprising even It is contacted in the solution of continuous liquid phase.Support may include the primer group comprising at least the first primer and the second primer.Can for example it lead to It crosses to the covalent linkage of support primer group is fixed on the support.In some embodiments, nucleic acid-templated comprising close The primer binding sequence of target sequence.Primer binding sequence can be complementary to the sequence of the first primer and the sequence of optionally the second primer Column.Target sequence can not be complementary to the primer in primer group.In some embodiments, nucleic acid-templated primer binding sequence is miscellaneous It hands over to the first primer.Polymerase switching templates can be used to extend the first primer, to form the first primer of extension.The primer of template Binding sequence can at least partly separate (such as denaturation or unwinding) with the first primer of extension.It optionally carries out separating while tieing up Hold hybridizing between the part of template and the first primer of extension.The part of isolated primer binding sequence then can be hybridized to Two primers.Optionally, it carries out such hybridization while maintaining hybridizing between the other parts of template and the first primer of extension. Polymerase switching templates can be used to extend the second primer, to form the branch comprising the first primer extended and second primer of extension Hold object.The part of the extension of second primer of the first primer and/or extension of extension may include the sequence for being complementary to target sequence.

In some embodiments, present disclosure generally relates to clone on the support in amplified reaction solution The method of ground amplification of nucleic acid template, comprising: contacted nucleic acid-templated in liquid solution with support, wherein support includes packet The primer group of fixation containing at least the first primer and the second primer, and the wherein nucleic acid-templated primer knot comprising close to target sequence Sequence is closed, wherein primer binding sequence is complementary to the sequence of the first primer and the sequence of the second primer, and target sequence is not complementary Primer in primer group；Nucleic acid-templated primer binding sequence is hybridized to the first primer；Extended using polymerase switching templates The first primer, to form the first primer of extension；By at least partly drawing with the first of extension for the primer binding sequence of template Object denaturation, while maintaining hybridizing between another part of template and the first primer of extension；By the primer binding sequence of denaturation Partial hybridization to the second primer, while maintaining hybridizing between the other parts of template and the first primer of extension；With use Polymerase switching templates extend the second primer, so that the support comprising the first primer extended and second primer of extension is formed, The extension of the first primer and the second primer extended that wherein extend respectively contains the sequence for being complementary to target sequence.Primer group can wrap Containing substantially the same primer, the substantially the same primer has not more than 1,2,3,4 or 5 nucleotide not in sequence Together.In some embodiments, primer group includes different primer, and at least some of the primer include to be complementary to drawing for template The sequence of object binding sequence.In some embodiments, the primer of primer group is not complementary to 5 ' half edge sequences of end of template.One In a little embodiments, the primer of primer group is not complementary to 3 ' half edge sequences of end of the primer of any extension of support.Some In embodiment, the primer of primer group is not complementary to any sequence in addition to primer binding sequence of template.

In some embodiments, present disclosure generally relates in amplified reaction solution in support group on gram The method of grand ground amplification of nucleic acid template group, comprising: according to any method disclosed herein in the first core on the first support Amplification the first template in clone ground on acid template, and expand the second nucleic acid mould with cloning on the second support according to identical method Plate, wherein all supports are included in single Continuous Liquid Phase during amplification.

Among other things, the side for generating the clone group of the limitation of clonal expansion of fixation of single-stranded template sequence is provided Method, comprising: single-stranded template sequence (" template 1 ") is connected to fixed site (" IS1 ") by (a), and wherein IS1 includes multiple copies It can be substantially hybridized to the primer (" IS1 primer ") of the fixation of template 1, and template 1 is bound to and with IS1 primer hybridization IS1, and (b) template 1 is expanded in the solution using IS1 primer and revocable primer (" SP1 primer "), wherein being complementary to single-stranded The chain of the amplification of template 1 cannot substantially hybridize when to be single-stranded with the primer on IS1, wherein expanding rising in template 1 to IS1 The hybridization point surrounding that begins generates the clone group of the limitation of fixed clonal expansion.

Additionally provide the separation and fixation for generating the first template sequence (" template 1 ") and the second template sequence (" template 2 ") Clone group method, the method includes the first and second template sequences of amplification to generate substantially to be connected to the first fixed bit The clonal expansion subgroup of the template 1 of point (" IS1 ") rather than second fixed site (" IS2 "), or be substantially connected to IS2 rather than The clonal expansion subgroup of the template 2 of IS1, in which: (a) two templates and all amplicons are all contained in identical Continuous Liquid Phase It is interior, wherein Continuous Liquid Phase and first and second fixed site (respectively, " IS1 " and " IS2 ") contacts, and wherein IS1 and IS2 It is spatially separated, (b) template 1 includes the first subsequence (" T1-FOR ") and at it an end when for single stranded form Opposing end portions include the second subsequence (" T1-REV "), and (c) template 2 includes the first sub- sequence an end when for single stranded form It arranges (" T2-FOR ") and includes the second subsequence (" T2-REV ") in its opposing end portions, (d) IS1 includes the fixation of multiple copies Nucleic acid primer (" IS1 primer "), the primer can substantially be hybridized to T1-FOR and T2-FOR when T1 and T2 is single-stranded, (e) IS2 includes the primer (" IS2 primer ") of the fixation of multiple copies, and the primer can be substantially hybridized to when T1 and T2 is single-stranded T1-FOR and T2-FOR, (f) reverse complement of T1-REV cannot substantially be hybridized to the primer on IS1 when to be single-stranded, but Revocable primer (" SP1 ") in Continuous Liquid Phase can be substantially hybridized to；(g) reverse complement of T2-REV is when to be single-stranded The primer that cannot be substantially hybridized on IS2, but can substantially be hybridized to revocable primer (" SP2 ") in Continuous Liquid Phase

Optionally, in any method being described herein, it is any being capable of base from the nucleic acid that a fixed site is dissociated Nucleic acid the appointing to another fixation site of described two fixed sites and the dissociation described in Continuous Liquid Phase is hybridized in sheet What mobile (such as passing through diffusion, the movement of convection current) is substantially unobstructed.

Optionally, in any method being described herein, Continuous Liquid Phase is contacted with IS1 and IS2 simultaneously.

Optionally, in any method being described herein, the first part for the template that fixed primer combines not with The second part of template is overlapped, and the complement of the second part is combined by revocable primer.

Optionally, in any method being described herein, at least one template to be amplified is being incited somebody to action from input nucleic acid It is generated after nucleic acid and at least one fixed bit point contact.

Optionally, any method described herein include the following steps: (a) by the support comprising fixed primer with Single stranded nucleic acid template contact, in which: by the primer binding sequence (PBS) on the first fixed primer hybridization to template；(b) with mould Plate dependence extends to extend the first primer of hybridization to form the chain of extension, and the chain of the extension is complementary to template and at least Partial hybridization is in template；(c) from the complementary strand partial denaturation template of extension so as at least partially single-stranded the form (" trip of PBS From part ")；(d) free fraction is hybridized to the second primer do not extend, fixed；(e) extended with Template Dependent extension Second primer is to form the chain of the extension for being complementary to template；(f) optionally, the nucleic acid chains of the fixation of the extension of annealing are divided each other From.

Optionally, in any method being described herein, (a) forms nucleic acid double chain during amplification to comprising rising Beginning template and/or the chain of amplification；The double-strand does not suffer from the complete denaturation for the double-strand that will lead to substantial amounts during amplification Condition.

Optionally, in any method being described herein, by obtaining multiple input double-strands to be amplified or single-stranded Nucleic acid sequence (sequence can be known or unknown) and addition or generation on the end of at least one input nucleic acid First universal linker sequence and the second universal linker sequence generate single-stranded template；Wherein the first universal linker sequence hybridization To the reverse complement of IS1 primer and/or IS2 primer, and second universal linker sequence, to be hybridized at least one non-solid Fixed primer.Connector can be double-strand or single-stranded.

Optionally, in any method being described herein, in the first and second ends of the single-stranded template sequence First and second nucleic acid linker sequences are provided.

Optionally, in any method being described herein, also by label be added to one or more nucleic acid sequences (such as Template or primer or amplicon), the label makes it possible to identify the nucleic acid comprising label.

Optionally, in any method being described herein, all draw what at least one fixed on site or support Object sequence having the same.Optionally, fixed site or support include multiple primers at least two different sequences. In some embodiments, fixed site or support include at least one target specific primer.

Optionally, in any method being described herein, continuous media is flowable.Optionally, revocable core Acid molecule is blended in Continuous Liquid Phase during at least part of amplification procedure, such as described herein at any one or more The step of or circulation during, be substantially uncrossed.

Optionally, in any method being described herein, in the period of during amplification in mixing be not to be obstructed substantially Hinder.For example, mixing is substantially uncrossed during the entire duration of amplification.

In embodiments, using RPA, that is, recombinase-polymeric enzymatic amplification (see, for example, WO2003072805, by drawing With being incorporated herein) realize amplification.Optionally RPA is carried out in the case where no temperature or the substantial variation of reagent conditions. In embodiments herein, recombinase and/or single strand binding protein can be used to realize partial denaturation and/or amplification, including Any one or more steps or method described herein.It is optionally combined with single strand binding protein (SSB), recombinase appropriate Including RecA and its protokaryon or eukaryon analog or its functional fragment or variant.In embodiments, recombinase agent is optionally Coating single stranded DNA (ssDNA) such as amplimer has same to form nucleoprotein filament chain in the nucleoprotein filament chain intrusion template The double stranded region of source property.This optionally generates short hybrid and alternative chain bubble (displaced strand bubble) (is referred to as D- Ring).In embodiments, the free 3 '-end of silk chain is extended through archaeal dna polymerase to synthesize new complementation in D- ring Chain.Complementary strand replaces the marriage chain of the original pair of template in extension.In embodiments, one or more amplimers pair It is contacted with before the template contacts of optionally double-strand with one or more recombinase agent.

In any method being described herein, the amplification of template (target sequence) includes by recombinase agent and at least one expansion Increase one or more contacts of primer pair, to form one or more " forward direction " and/or " reversed " RPA primers.Optionally go Except any not recombinase agent with the association of one or more primers.Optionally, then by one or more forward direction RPA primers and mould The contact of plate chain, the template strand optionally have the region at least one RPA Primers complementary.It can be by template strand to RPA The contact position of primer and complementary template, the contact optionally lead to hybridizing between the primer and template.Optionally, it utilizes One or more polymerases (such as in the presence of dNTP) are along 3 ' ends of template extension primer to generate double-strandednucleic acid With displacement template strand.Amplified reaction may include such contact and the repetitive cycling extended until can get desired amplification journey Degree.Optionally, the displacement chain of amplification of nucleic acid is reacted by parallel RPA.Optionally, the displacement chain of nucleic acid by by its successively with One or more complementary primer contacts are to expand；(b) complementary primer is extended by any strategy described herein.

In embodiments, one or more primers include " forward direction " primer and " reversed " primer.By two kinds of primers and mould Plate contact optionally leads to the first duplex structure in the first part of first chain and leads in the second part of second chain Cause duplex structure.Optionally, extend positive and/or reverse primer 3 ' ends with one or more polymerases to generate the first He First and second displacement chains of the second double-strandednucleic acid and nucleic acid.Optionally, the second displacement chain it is at least partly complementary to each other and It can hybridize to form sub- double-strandednucleic acid, double stranded template nucleic acid can be used as in subsequent amplification cycles.

Optionally described first and described second, which replaces chain, is at least partially complementary to the described first or described second primer simultaneously And the described first or described second primer can be hybridized to.

In the alternative embodiment of any method or step or composition or array that are described herein, support is optional Ground includes the primer of the fixation with more than one sequence.After template nucleic acid chain is hybridized to the first complementary immobilized primer, then It may extend away the first primer and template and primer can partially or even wholly be separated to each other.The primer of extension can then be moved back Fire may extend away the second primer extremely with the second immobilized primer of the sequence different from the first primer.Then separable (such as that This is completely or partially denaturalized) two kinds of primers extended and it can be used as in turn to the mould for being used to extend other immobilized primer Plate.To provide amplification, the fixed nucleic acid molecules of repeatable process.In embodiments, which leads to difference there are two types of tools Sequence complimentary to one another fixation primer extension product, wherein all primer extension products are fixed to support in 5 ' ends Object.

In some embodiments, disclosed method includes amplification, wherein amplification includes that chain is overturn.It is described below In " overturning " embodiment, extend two or more primers to form the chain of two or more corresponding extensions.Optionally, Two or more extended primers include substantially the same sequence or consisting essentially of, and corresponding extension The part of the extension of chain is at least partly different and/or complimentary to one another.

One exemplary implementation scheme of overturning is as described below.Such as walked by template and expand starting template, to generate The chain (for convenience of rising, seeing that it will be referred to as " forward direction " chain) of multiple primer-extensions.Optionally, positive chain is complementary to starting template. Optionally, positive chain is fixed on the support.Optionally, positive chain includes substantially the same sequence, such as positive chain is basic It is upper mutually the same.In embodiments, by extend one or more fixed primers (" forward direction " primer) on the support come Form positive chain.Forward primer and/or positive chain are optionally connected to support in its 5 ' end or nearby.Optionally, primer- The one or more of the positive chain of extension includes 3 ' sequences being referred to as from hybridization sequences, and the 3 ' sequence is not present in not extending Primer in and 5 ' sequences can be hybridized under selected conditions (this process will be referred to as " from hybridize ").5 ' sequences are optionally It is the part for the forward primer not extended.In instances, positive extension products form " stem-loop " structure after such hybridization. Optionally, the forward primer not extended is in its 3 ' end or nearby comprising " cleavable " nucleotide susceptible to cutting.In reality It applies in scheme, cleavable nucleotide is connected at least one other nucleotide, institute by connecting between " scissile " nucleosides It states and connects and can be cut under conditions of will not substantially cut phosphodiester bond between nucleosides.

After extension, optionally permission forward direction-primer extension product (i.e. positive chain) hybridizes certainly.In other embodiments, Allowing from after hybridizing, in cleavable nucleotide (such as the nucleotide for forming scissile connection with adjacent nucleotide) Scissile junction cut positive chain.Cutting leads to two segments of primer-extension products (the positive chain extended).? In embodiment, the first segment include the original forward primer not extended at least partly.Optionally, the first segment does not include The sequence of any extension.Optionally, the first segment is fixed (for example, because the forward primer not extended has been fixed ).In embodiments, the second segment includes the sequence extended.Optionally, the second segment includes that the primer not extended can cut Any 3 ' part except the nucleotide cut or any part not comprising the primer not extended.Optionally, the second segment passes through It is hybridized to first part from hybridization sequences.

In instances, cleavable nucleotide is the nucleotide removed by one or more enzymes.Enzyme may, for example, be glycosyl Change enzyme.According to N- glycosylase activity, cleavable nucleotide is optionally discharged from double-stranded DNA for glycosylase.Optionally, may be used The removal of the nucleotide of cutting is generated without base, without purine or without pyrimidine site.Optionally for example pass through another enzymatic activity Further to modify abasic site.Optionally, abasic site is modified to generate base notch by lyase.Lyase example Such as cut the 3 ' and/or 5 ' of abasic site.Optionally occur to cause to remove nothing in 5 ' and 3 ' ends by the cutting of lyase Base position and leave base notch.Exemplary cleavable nucleotide such as 5- hydroxyl-uracil, 7,8- dihydro -8- hydroxyl bird Purine (8- hydroxyl guanine), 8- hydroxyl adenine, fapy- guanine, methyl-fapy- guanine, fapy- adenine, aspergillus flavus poison Plain B1-fapy- guanine, 5- hydroxy-cytosine can be identified and be gone by a variety of glycosylases divided by formation without purine site.One The suitable enzyme of kind is formamido group pyrimidine [fapy]-DNA glycosylase, also referred to as 8- hydroxyl guanine DNA glycosylase or FPG. FPG can be used as N- glycosylase and AP- lyase.N- glycosylase activity optionally discharges impaired purine from double-stranded DNA, To generate without purine (AP site), wherein phosphodiester backbone is optionally complete.AP- lyase activity cuts AP site 3 ' and 5 ' AP site and leave single base notch to remove.In instances, cleavable nucleotide is 8- hydroxyl adenine, Single base notch is converted to by the FPG with glycosylase and lyase activity.

In another embodiment, cleavable nucleotide is uridine.Optionally, uridine is cut by " USER " reagent, The reagent includes uracil dna glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII, wherein UDG It is catalyzed the excision of uracil base, is formed and keeps the complete of phosphodiester backbone, and its simultaneously without base (no pyrimidine) site In cut the lyase activity of nuclease VIII and destroy phosphodiester backbone in the 3 ' of abasic site and 5 ' sides to discharge alkali-free The deoxyribose of base is subsequently optionally converted the phosphate group on 3 ' ends of cleaved products to-OH base using kinases.

Optionally at least one segment cut is contacted with polymerase.Optionally first fixed segment can be by polymerase Extend.If it is expected in this way, the segment of the second hybridization can be used as the template of the extension of the first segment.In embodiments, shape At " overturning " double-strand extension products.The product of the overturning optionally by it is described herein it is any in a manner of undergo template row It walks.When overturning and unturned experience template walking, two different extension products groups are formed, two of them, which extends, to be produced Object part having the same (corresponding to the primer not extended) and the part complimentary to one another (portion of the extension corresponding to extension products Point).

In embodiments, reagent is optionally being extended by the positive chain and single-stranded " montage " joint sequence that will extend It is contacted in the presence of (such as polymerase and dNTP) by aim sequence for example from hybridization sequences or new primer knot Position addition is closed in 3 ' ends of the positive chain of extension.The montage sequence optionally includes the positive chain for being essentially complementary extension 3 ' end sections 3 ' partially and be essentially complementary 5 ' parts of aim sequence to be added.Hybridize by montage connector To 3 ' ends of the positive chain of extension, montage connector is used to prolong positive chain experience template dependent polymerase as template It stretches.Such extension causes aim sequence to the addition of 3 ' ends of the positive chain extended.

Therefore, primer extend described herein and/or any method of amplification may include either one or two of following steps or Multiple: the positive chain for extending fixed forward primer to generate multiple extensions of (a) being walked by template, the forward direction chain is optionally It is identical；(b) optionally using montage connector be hybridized to extension positive chain 3 ' ends and use montage connector as template Extend positive chain experience template dependent polymerase, so that other 3 ' sequences are added to the positive chain further extended, The part of 3 ' sequences of middle addition be complementary to the forward primer not extended part and be hybridized to it to form stem ring knot Structure；(c) cleavable at or near the tie point of positive chain-ordering for being located at the forward primer sequence and extension that do not extend Cut positive chain in the easy cutting junction of nucleotide；Cleavable nucleotide is optionally removed, to generate two cuttings Segment, wherein the first segment includes the portion for the forward primer of the 3 ' primers-complementary series being hybridized in the second segment not extended Point；(d) the second segment is optionally used to extend the first segment experience polymerase to generate the positive chain of overturning as template； (e) the second montage connector is optionally hybridized to 3 ' ends of the positive chain of overturning, and montage connector is used to make just as template Extend to chain experience Template Dependent, so that other 3 ' sequences are added to the positive chain of overturning, wherein the 3 ' sequences added Part be not present in overturning chain in new primer binding sequence；(f) optionally through contacted with new primer and with Any method (such as described in this article) extends or expands to extend or expand turning over comprising new primer binding sequence The chain turned.New primer will not be incorporated into the chain of unturned chain or the overturning not being extended still further in step (e).

Fig. 8 shows that the schematic diagram of exemplary chain overturning and Running strategy describes.(A) template is walked, and (B) chain is overturn to produce The chain of raw overturning, (C) adds new primer binding sequence Pg ' on final overturning chain.

Optionally, by any amplification method of single support in this article, wherein single support, which has, to be hybridized In multiple primers of template.In such embodiments, the adjusting template before template gleanings and solid support into contact The concentration of gleanings is so that the individual template molecule in gleanings is at least 10²、10³、10⁴、10⁵、4x 10⁵、5x 10⁵、6x 10⁵、8x 10⁵、10⁶、5x 10⁶Or 10⁷A molecule/mm²Density be connected or association (such as is fixed on solid by being hybridized to Primer on support).

Optionally, in situ on the support to expand individual template molecule, generation is spatially confined to the miscellaneous of starting template Clone group around intersection point.Optionally, it expands from the template individually expanded and generates no more than about 10²、10³、10⁴、10⁵、10⁶、 10⁷、10⁸、10⁹,10¹⁰、10¹¹、10¹²、10¹⁵Or 10²⁰Amplicon.Optionally, the colony of clonal expansion is at least 10²、 10³、10⁴、10⁵、4x 10⁵、5x 10⁵、6x 10⁵、8x 10⁵、10⁶、5x 10⁶Or 10⁷A molecule/mm²Density be located at solid branch It holds on object.

It in some embodiments, can multiple nucleic acid be bound to wherein with one or more supports by nucleic acid gleanings It is contacted under conditions of identical support.Such contact can be particularly used for being related to nucleic acid in the difference of identical support The method of parallel clonal expansion in region.The adjustable nucleic acid quantity ratio long-pending to support surface, for example, by ensuring Nucleic acid is being properly spaced in support to promote the monoclonal group of the nucleic acid expanded substantially between different clone groups It is formed in the case where no cross contamination to promote monoclonal to be formed.Such as when using single support, by core to be amplified The gleanings of acid are adjusted to such dilution so that the clone group of the resulting amplification generated from individual nucleic acid does not connect generally It is continuous or separation such as non-overlapping.For example, the individual in the clone group of 50%, 70%, 80% or 90% or more amplification Do not have to spread the nucleic acid being not substantially identical in nucleic acid.Optionally, different amplification groups does not contact or complete with others amplification groups Full weight is folded or the detection method of usable selection is distinguished from each other.

In some embodiments, nucleic acid is connected to the surface of support.In some embodiments, nucleic acid can adhere to In support.For example, for the support comprising hydrogel or other porous matrixes, nucleic acid can be attached to the whole of support A volume includes on surface and in support.

In some embodiments, support (or at least one support in support group) can be connected at least one A primer is optionally connected to primer group.For example, support (or at least one support) may include primer group.Primer group's Primer can be being substantially identical or may include substantially the same sequence.One of primer, some or all can wrap Containing the sequence for being complementary to one or more nucleic acid-templated interior sequences.In some embodiments, primer group may include at least two The primer of a incomplementarity.

Primer can be connected to support by its 5 ' end, and have 3 ' free ends.Support can be glass slide Surface or globule surface.Primer has low melting temperature, such as few (dT)₂₀, and the low of gleanings connector can be hybridized to T_mRegion.Distance between primer needs shorter than joint length to allow template to walk, or selectively, has 5 ' end lengthening joints Long primer will increase walking chance.

In some embodiments, by support and primer and template (or reverse strand), primer and template are miscellaneous each other wherein Friendship is attached and/or contacts under conditions of nucleic acid double chain to be formed.Double-strand may include the complementary series comprising template and primer Double stranded section, wherein at least one nucleotide residue of complementary series is base pairing each other.In some embodiments, Double-strand also may include single stranded portion.Double-strand also may include single stranded portion.Single stranded portion may include any in template (or primer) It is not complementary to the sequence of any other sequence in primer (or template).

The non-restrictive illustrative method for the nucleic acid amplification cloned on the support is as described below.Clone ground on the support Amplification of nucleic acid (for convenience, will be referred to as reverse strand), and the complementary forward direction that multiple copies are connected on the support is drawn Object.Exemplary nucleic acid is one of multiple DNA gleanings molecules, such as multiple nucleic acid members have in its 5 ' and/or 3 ' end The sequence of one or more common (" connectors ") simultaneously has variable sequence, such as gDNA or cDNA therebetween.In embodiment In, 3 ' common grounds such as connector has respirable (such as low T_m) region, and 5 ' consensus (such as connector) are optional Ground has less respirable (such as higher T_m) region, or vice versa.In another embodiment, 5 ' and 3 ' is common Sequence is all respirable.Respirable (such as low T_m) region is, for example, to be rich in the region of A, T and/or U, such as rich in AT The sequence of (or U), such as polyT, polyA, polyU and A, T and U base or it is complementary to any group of base of such base It closes.Illustrative methods describe in this article.

One non-restrictive illustrative method of the nucleic acid amplification of the clone carried out on the support by " template walking " It is shown in Fig. 1.The non restrictive description of the illustrative methods of template walking is as described below.

Double-stranded DNA library molecule is denaturalized and single stranded DNA is connected to support by the primer being hybridized on support Object.DNA molecular quantity is provided to the ratio of support area or globule quantity monoclonal is promoted to be formed.

Primer 5 ' is connected on primer by it and has free 3 '.Support can be surface or the globule of glass slide Surface.Primer has low melting temperature, such as few (dT)₂₀Or it is few (dA)₃₀, and the low T of library connector can be hybridized to_mArea Domain.Distance between primer can be shorter than joint length to allow template to walk, or selectively, the length with 5 ' end lengthening joints Primer will increase the chance of walking.

Ground amplification of nucleic acid is cloned on the support, and the primer of multiple copies is connected on the support.Exemplary nucleic acid It is one of multiple DNA library molecules, the multiple DNA library molecule for example has in its 5 ' and/or 3 ' end a kind of or more The sequence of kind common (such as " connector ") simultaneously has variable sequence, such as gDNA or cDNA therebetween.In embodiments, 3 ' Connector has low T_mRegion, and 5 ' connectors T optionally with higher_mRegion, or vice versa.Low T_mRegion is, for example, richness Region containing pyrimidine, such as the sequence rich in AT (or U), such as polyT, polyA, polyU and A, T and U base or be complementary to Any combination of the base of such base.Illustrative methods describe in this article.

Using DNA polymerization or extension mixture, optionally include any one or more reagents such as enzyme, dNTP and Buffer incubates one or more primers, and no matter it is dissolved form or is connected to support.Extension primer (such as just To primer).Optionally, extension is that primer extends along the Template Dependent of template, including the continuous kernel being respectively complementary in template The nucleotide of thuja acid is continuously incorporated to, and (is also referred to as reversely put down so that forward primer that is extension or not extending is complementary to reverse strand It is capable or complementary).Optionally, by extending with polymerase activity or other active enzyme such as polymerases of extension to realize.Enzyme Optionally having other activity includes 3 ' -5 ' exonuclease activities (proofreading activity) and/or 5 ' -3 ' exonuclease enzyme activity Property.Alternatively, in some embodiments, it is active one or more that enzyme can lack these.In embodiments, polymerase has Strand-displacement activity.The example of useful strand displacement polymerase includes bacteriophage Ф 29DNA polymerase and Bst DNA polymerization Enzyme.Optionally, enzyme is in raised temperature, for example, at 45 DEG C or on, 50 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or 85 DEG C it It is upper active.

Exemplary polymerases are Bst archaeal dna polymerase (exonuclease are negative), are 67kDa bacillus stearothermophilus (Bacillus stearothermophilus) archaeal dna polymerase albumen (large fragment) (being illustrated in accession number 2BDP_A), has 5 ' -3 ' polymerase activities and strand-displacement activity list lack 3 ' -5 ' exonuclease activities.Other polymerases include from aquatic The Taq DNA polymerase i (being illustrated in accession number 1TAQ) of Thermus (Thermus aquaticus) comes from Escherichia coli The Eco DNA polymerase i (accession number P00582) of (Escherichia coli) comes from hyperthermophile (Aquifex Aeolicus Aea DNA polymerase i (accession number 067779) or its functional fragment or variant), such as in nucleotide level Above there is the functional fragment or variant of at least 80%, 85%, 90%, 95% or 99% sequence identity.

Normally, extend step and generate nucleic acid, it includes the double-stranded duplex parts that two of them complementary strand hybridizes each other. In one embodiment, walking includes that nucleic acid is made to undergo partial denaturation condition, the partial denaturation condition denaturing nucleic acid chain Part but it is not enough to fully denaturing nucleic acid over the whole length.In embodiments, it is expert at the part or whole of walking program Nucleic acid is not set to undergo complete Denaturing during duration.

In embodiments, design in this way the sequence of minus strand and/or normal chain so as to primer binding sequence or part thereof be can Breathing, i.e., it is susceptible to being denaturalized under the condition of selection (such as amplification condition).Respirable part is optionally than most of phase The nucleic acid with random sequence like length is more susceptible, or than comprising can at least another part of chain of respiration sequence be easier to Sense.Optionally, can respiration sequence shown under the amplification condition of selection significant quantity denaturation (for example, at least 10%, 20%, 30%, 50%, 70%, 80%, 90% or 95% molecule can be denaturalized completely in respiration sequence).Such as it can breathe Sequence design be under the condition (such as amplification condition) of selection 30,35,40,42,45,50,55,60,65 or 70 DEG C It is denaturalized completely in 50% chain molecule.

When by heating or raised temperature to realize partial denaturation, can illustratively breathe PBS be can be rich in phonetic (such as A and/or T and/or U with high-content) of pyridine.PBS is including, for example, poly-A, poly-T or poly-U sequence or more Poly- pyrimidine track.Optionally one or more amplifications or other primers (such as fixed primer) are designed as accordingly being complementary to this A little primer binding sequences.The exemplary PBS of nucleic acid chains includes poly-T sequence, for example, at least 10,15,20,25 or 30 thymidines The section of nucleotide, and corresponding primer has the complementary series of PBS, for example, at least 10,15,20,25 or 30 adenosine nucleosides The section of acid.Exemplary low melting point primer optionally have such high proportion (for example, at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) nucleobase: it usually (such as is being selected when primer hybridizes with complementary template Under the amplification condition selected) and complementary base formation not more than two hydrogen bonds.The example of such nucleobase includes A (adenine), T (thymidine) and U (uracil).Exemplary low melting point primer optionally has a high proportion of A (adenine), T (thymidine) And/or U's (uracil) is any one or more or derivatives thereof.In embodiments, (gland is fast comprising being complementary to A for derivative Purine), the nucleobase of T (thymidine) and/or U (uracil).The part for being hybridized to the primer of PBS optionally has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% A (adenine), T (thymidine) or U (uracil) nucleotide or any combination thereof.In another example, the part for being hybridized to the primer of PBS includes polyA sequence (for example, at least 5,10,15,20,25 or 30 nucleotide are long).Other Exemplary primers include (NA_x)_nRepetitive sequence.Optionally Ground, n (lowercase) are 2-30, such as 3-10, such as 4-8." N " is (uppercase) for any nucleotide-and optional Ground, N are C or G." A " be adenine abbreviated notation, " x " indicate in repetitive sequence adenine residue quantity, such as 2,3, 4,5,6,10 or more.Exemplary primers include (CAA)_n、CA)_n、(CAAA)_nOr even (GAA)_nMultiple repetitive sequences.

Optionally only a chain (such as chain forward or backwards) has respirable PBS.In another embodiment, just To and reverse strand all there is respirable PBS.Respirable PBS, which is optionally complementary to, is fixed to support or revocable (example Such as dissolved form) primer.Optionally, the chain comprising respirable PBS is fixed to support or revocable (such as dissolves Form).Optionally two kinds of primers are fixed or two chains are fixed.Optionally primer is not fixed, or Chain is not fixed.

Amplification cycles optionally include breathing (breathing), annealing and extend.Optionally pass through nucleic acid to be amplified Go through at least one these step be suitble to or optimal conditions.In embodiments, make nucleic acid experience suitable for more In the condition of these step (for example, anneal and extend, or breathing and extension).In some instances, all three these Step can occur simultaneously at identical conditions.

In illustrative methods, nucleic acid experience can be made to allow or promote the condition of breathing.In embodiments, when double-strand is double Two chains of conveyor screw are when substantially hybridizing each other but being denaturation in purpose Part portions, then it is assumed that " breathing " generation. What the respirable sequence (such as positive and/or reversed PBS with the low part Tm) of one or more of nucleic acid was hybridized to from it First complementary strand (such as chain forward or backwards) partial denaturization (" breathing "), so as to for being hybridized to another second chain.Example The first chain of property is the primer extension product from the first primer.Exemplary second chain be, for example, second do not extend primer (for example, PBS complementary oligonucleotide including, for example, dT or dA sequence).Optionally, the first and second chains are fixed on the support, and can To be (for example, sufficiently close adjacent to allow to walk) closely placed.Condition for breathing is optionally partial denaturation Condition, PBS is substantially denaturalized under the described conditions but another part of nucleic acid keeps hybridization or double-stranded state.Optionally, DNA Unwindase may include in the reactive mixture to promote partial denaturation.

Optionally, then make nucleic acid experience promote annealing condition, such as reduce temperature so that respirable PBS with It can hybridize between second chain.In embodiments, using identical condition to promote to breathe and extend.In another embodiment party In case, annealing conditions are different from breathing condition-for example, annealing conditions are non-Denaturings or promote the change for being less than breathing condition The condition of property.In instances, annealing conditions include temperature more lower than breathing condition (such as 37 DEG C), in the breathing condition Higher temperature (such as 60-65 DEG C) is used.Optionally, in one or more amplification cycles (such as most of amplification cycles Or essentially all of amplification cycles) during avoid complete Denaturing.

Optionally, one or more PBS- breathing and primer extension procedures are repeated as many times to expand initial nucleic acid.When one When kind or multiple nucleic acids reagent (such as primer) are fixed to support, primer-extension products before amplification for example due to not prolonging The primer for the extension stretched is kept substantially to the connection of support or and with such hybridizing for primer to the company of support It connects.

Optionally, the sample of the one or more nucleic acid groups to be amplified of preparation.Nucleic acid group can be single-stranded or double-stranded shape Formula；Optionally one or more nucleic acid respectively contain the nucleic acid chains with known 3 ' end sequence and known 5 ' end sequence, institute It is substantially the same or complementary that end sequence, which is stated, with one or more primers for amplification.It the 3 ' of nucleic acid chains partially can example Such as with fixed Primers complementary, and 5 ' partially can be identical as the primer of dissolution.5 ' and/or 3 ' between intragroup individual nucleic acid Part can be common (" general ") or constant.Optionally, intragroup nucleic acid respectively contains between common ground Different (such as unknown) sequences, such as genomic DNA, cDNA, mRNA, pairing (mate-pair) segment, exon group Deng.Gleanings can for example ensure to cover with enough members the corresponding hereditary source higher than 50%, 70% or 90%.

In some embodiments, what present disclosure generally related to nucleic acid amplification includes for the anti-of nucleic acid amplification Answer the composition and relevant system, device, kit and method of mixture.

In some embodiments, present disclosure generally relates to the combination comprising reaction mixture of nucleic acid amplification Object and relevant system, device, kit and method, the reaction mixture include Continuous Liquid Phase, the Continuous Liquid Phase packet Containing (i) polymerase and (ii) multiple supports, at least one of support is connected to the nucleic acid group of substantially monoclonal.

In some embodiments, present disclosure generally relates to the combination comprising reaction mixture of nucleic acid amplification Object and relevant system, device, kit and method, the reaction mixture include Continuous Liquid Phase, the Continuous Liquid Phase packet It include multiple supports of the first support and the second support containing (i) polymerase and (ii).

In some embodiments, present disclosure generally relate to nucleic acid amplification composition (and relevant system, Device, kit and method), it includes the reaction mixture comprising Continuous Liquid Phase, the Continuous Liquid Phase includes that (i) includes the Multiple supports of one support and the second support, (ii) include the multiple and different of the first polynucleotides and the second polynucleotides Polynucleotides and (iii) be used for isothermal nucleic acid amplification reagent.In some embodiments, for the reagent packet of nucleic acid amplification Nucleotide (such as multiple nucleotide) containing polymerase and one or more types.Optionally, for the examination of isothermal nucleic acid amplification Agent includes recombinase.

Optionally, the first and second polynucleotides have different sequences.

Optionally, at least one end of at least one of multiple and different polynucleotides is connected at least one few nucleosides Sour connector.

Optionally, at least one at least some of end of multiple and different polynucleotides includes common sequence.

Optionally, at least two of polynucleotides different in reaction mixture include common sequence.

Optionally, the first and second polynucleotides are different.

In some embodiments, liquid phase include comprising primer it is multiple in one or more supports.

Optionally, reaction mixture includes Continuous Liquid Phase.

Optionally, reaction mixture can be used to carry out isothermal or thermal cycle nucleic acid amplification.

Optionally, Continuous Liquid Phase includes either one or two of (i) one or more polymerases and/or (ii) at least one support Or any combination.

Optionally, Continuous Liquid Phase includes multiple supports.

Optionally, Continuous Liquid Phase includes the first support.

Optionally, Continuous Liquid Phase includes the second support.

Optionally, can will be multiple at least one support be connected to the nucleic acid group of substantially monoclonal.

Optionally, the first support can be connected to the nucleic acid group of the first substantially monoclonal.

Optionally, the second support can be connected to the nucleic acid group of the second substantially monoclonal.

Optionally, the nucleic acid group of the first and second substantially monoclonals includes different sequences or substantially the same sequence Column.

Optionally, first and second substantially monoclonals nucleic acid group under stringent hybridization conditions each other hybridization or it is not miscellaneous It hands over.

Optionally, the nucleic acid group of the first and second substantially monoclonals is different.

Optionally, the nucleic acid group of the first and second substantially monoclonals is non-complementary.

Optionally, reaction mixture includes the nucleotide for not carrying out exogenous marker.For example, nucleotide can be it is naturally occurring Nucleotide, or synthetic analogues of the optically detectable label not comprising fluorescence part, dyestuff or other external sources.

Optionally, reaction mixture is included in single reaction vessel.

Optionally, reaction mixture includes isothermal or thermal cycle reaction mixture.

Optionally, multiple supports include the inner wall of globule, particle, particle, sphere, gel, filter or pipe.

Optionally, can will be multiple at least one support be connected to multiple nucleic acid.

Optionally, can will be multiple at least one support be connected to one or more primers.Primer can be identical It is (or including common sequence) or different.

Optionally, at least one support can be connected to multiple the first primers.

Optionally, at least one support can be connected to multiple the first primers and multiple second primers.

Optionally, multiple the first primers include substantially the same sequence.

Optionally, multiple the first primers include at least one include with the polynucleotides of multiple and different polynucleotides extremely The first primer of the identical or complementary sequence of small part.

Optionally, multiple second primers include at least one include with the polynucleotides of multiple and different polynucleotides extremely Second primer of the identical or complementary sequence of small part.

In some embodiments, at least one polynucleotides of multiple and different polynucleotides include in the first primer Sequence is substantially the same or the First ray that is substantially complementary.In some embodiments, at least one polynucleotides also wraps Containing the second sequence that is substantially the same with the sequence in the second primer or being substantially complementary.In some embodiments, it is multiple not Essentially all polynucleotides in same polynucleotides include First ray and the second sequence.

Optionally, at least one support in multiple is connected to 2-10 different multiple primers.

Optionally, 2-10 different multiple primers include different sequences.

Optionally, 2-10 different multiple primers include at least partly progress of at least one and different polynucleotides The sequence of hybridization.

Optionally, 2-10 different multiple primers include at least one and the consensus in different polynucleotides The sequence at least partly hybridized.

Optionally, at least one of support is connected at least one unique identification sequence of barcodes.

Optionally, the nucleic acid group of the first and second substantially monoclonals has basically the same or different sequences.

Optionally, reaction mixture includes at least one recombinase.

Optionally, recombinase can be catalyzed homologous recombination, chain intrusion and/or D- ring and be formed.

Optionally, recombinase is the part of nucleoprotein filament, and the nucleoprotein filament includes to be connected in reaction mixture to adhere to In the recombinase of the primer of support.Support or in the solution can be connected to by the primer that recombinase connects.

Optionally, reaction mixture includes nucleoprotein complex or multiple nucleoprotein complexs.

Optionally, reaction mixture includes the first nucleoprotein complex.

Optionally, reaction mixture includes the second nucleoprotein complex.

Optionally, at least one nucleoprotein complex in multiple includes that at least one is connected to the recombinase of primer.

Optionally, reaction mixture includes to answer containing at least one first nucleoprotein for being connected to the recombinase of the first primer Close object.

Optionally, reaction mixture includes to answer containing at least one second nucleoprotein for being connected to the recombinase of the second primer Close object.

Optionally, recombinase includes to come from T4, T2, T6, Rb69, Aeh1, KVP40, acinetobacter (Acinetobacter) bacteriophage 133, Aeromonas (Aeromonas) bacteriophage 65, cyanophage P-SSM2, indigo plant Green alga bacteriophage PSSM4, cyanophage S-PM2, Rb14, Rb32, Aeromonas bacteriophage 25, vibrio (Vibrio) Bacteriophage nt-1, phi-1, Rb16, Rb43, bacteriophage 31, bacteriophage 44RR2.8t, Rb49, bacteriophage Rb3 or bacteriophage LZ2 Bacteriophage recombinase.

Optionally, recombinase includes the uvsX recombinase from T4 bacteriophage or the recA recombination from Escherichia coli Enzyme.

Optionally, reaction mixture also includes polymerase.

Optionally, it polymerize 5 ' to 3 ' exonuclease activity of azymia.

Optionally, polymerase includes strand-displacement activity.

Optionally, polymerase includes thermal stability or heat sensitivity polymerase.

Optionally, polymerase includes archaeal dna polymerase or RNA polymerase.

Optionally, reaction mixture also includes the nucleotide of at least one type.

Optionally, at least one support in multiple includes at least one primer.

Optionally, the first support is connected to the first substantially nucleic acid group of monoclonal and the second support and is connected to The nucleic acid group of two substantially monoclonals.

Optionally, the nucleic acid group of the first and second substantially monoclonals has different nucleic acid sequences.

Optionally, the nucleic acid group of the first and second substantially monoclonals does not hybridize each other under stringent hybridization conditions.

Optionally, the nucleic acid group of the first and second nucleic acid substantially monoclonal is not identical and incomplementarity.

Optionally, reaction mixture includes the polynucleotide template and/or (ii) at least one to be amplified of (i) at least two A nucleoprotein filament compound.

Optionally, reaction mixture includes that at least one is connected to polynucleotides, primer, template or the expansion of resistance compound Increase production object.As used herein, term " resistance compound " and its variant describe such any Chemical composition that: the combination Object can be connected to nucleic acid and hinder its diffusion by reaction mixture, but still allow in nucleic acid synthesis reaction using such Polynucleotides, primer, template or amplified production carry out nucleic acid synthesis.Such resistance compound is to nucleic acid in synthetic reaction It connects the mobility usually reduced such nucleic acid in the reactive mixture and can be used for preventing using identical reaction mixing The cross contamination of amplified production or template between the different synthetic reactions that object occurs.In some embodiments, resistance component The quantity or ratio of monoclonal product can be increased to the connection of one or more nucleic acid compositions.

In some embodiments, this introduction provides the method for being used for nucleic acid amplification, and the nucleic acid amplification includes at least one It is a to flow the nucleic acid (such as primer) sexually revised.In some embodiments, the nucleic acid sexually revised is flowed to show increased or subtract The small mobility by aqueous medium.In some embodiments, the nucleic acid of modification is included in along any position of length nucleic acid On be connected to nucleic acid (example of one or more change nucleic acid by the compound (such as resistance compound) of the mobility of aqueous medium Such as primer).In some embodiments, the resistance compound for changing the mobility of nucleic acid can be connected in nucleic acid amplification reaction Any primer, including first, second, third, fourth or any other primer.For example, can be in 5 ' ends, 3 ' ends and/or interior One or more resistance compounds are connected to nucleic acid on either one or two of portion position or any combination.In some embodiments, The nucleic acid of modification can be covalently or non-covalently connected to the resistance compound for changing the mobility that nucleic acid passes through aqueous medium.Example Such as, resistance compound, can be by changing the nucleic acid of modification compared to the nucleic acid for lacking the compound connected when being connected to nucleic acid Outer dimension, length, radius, shape or charge aqueous fluid dynamic drag is provided.In some embodiments, it is connected to core The resistance compound of acid can be changed the interaction between nucleic acid and aqueous medium and (compared to aqueous medium and lack the compound connected Nucleic acid between interaction).In some embodiments, resistance compound can be synthesis, recombination or natural deposit ?.In some embodiments, resistance compound can be with point, uncharged, polar or hydrophobic.Some In embodiment, resistance compound can be it is linear, branch or have dendroid paradigmatic structure (dendrimeric structure).In some embodiments, resistance compound may include the single part or poly- of nucleosides, sugar, rouge or amino acid Close object.

Optionally, resistance compound includes saccharide part, polysaccharide, albumen, glycoprotein or polypeptide.Optionally, resistance compound Including BSA, lysozyme, beta-actin, myosin, lactalbumin, ovalbumin, beta galactosidase, lactic dehydrogenase Or immunoglobulin (such as IgG).

Optionally, changing the resistance compound that nucleic acid passes through the mobility of aqueous medium includes one or more polyethylene glycol oxides (PEO) or the part polypropylene oxide (PPO), the polymer including polyethylene glycol oxide (PEO) or polypropylene oxide (PPO).In this way The non-limiting example of polymer include triblock copolymer (such as PEO-PPO-PEO), Pluronics^TMType polymer and Hydrophobically modified PEO polymer.Optionally resistance compound includes one or more amino acid moieties, polypeptide and cluster peptide. Optionally, resistance compound includes saccharide part, polysaccharide, hydrophobically modified polysaccharide, cellulose derivative, carboxymethyl cellulose Sodium, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose or hypromellose.Optionally, resistance compound packet Include solvable united (HASE) polymer of hydrophobically modified alkali, hydrophobically modified polyacrylamide, thermally sensitive polymer or N- N-isopropylacrylamide (NTPAAm), optionally, resistance compound include poly(ethylene glycol) methyl ether acrylate (PEGMEA), Tetraethylene glycol diacrylate (TEGDA), poly(ethylene glycol) dimethacrylate (EGDMA) or N, N '-- two-acryloyl of methylene Amine (NMBA).

Optionally, resistance compound includes albumen or polypeptide, including BSA, lysozyme, beta-actin, myosin, cream Albumin, ovalbumin, beta galactosidase or lactic dehydrogenase.In some embodiments, can be connected by amido or sulfydryl It connects and resistance compound is connected to nucleic acid.

In some embodiments, flow the nucleic acid that sexually revises include be connected to binding partners (such as with receptor portion The affinity part of split-phase interaction) nucleic acid.In some embodiments, acceptor portion can be used as resistance compound.One In a little embodiments, affinity can be partially attached to nucleic acid, and affinity part (it is used as resistance compound) and receptor Part interacts.For example, nucleic acid can be connected to the biotin moiety in combination with avidin sample part.Antibiotin Albumen sample part can be used as resistance compound.Avidin sample part includes avidin and avidin Can any derivative, analog and other unnatural forms in conjunction with biotin moiety.Other example packets of binding partners Include epitope (such as albumin A) and its respective antibody (such as anti-FLAG antibody) and fluorescein and anti-fluorescein antibody.This Field technical staff combines the other binding partners for being used to for resistance compound to be connected to nucleic acid are readily recognized.

Optionally, can by resistance compound and primer are connected to binding partners pair two members each by Resistance compound is connected to primer.

Optionally, at least one primer in reaction mixture includes biotin.

Optionally, resistance compound includes avidin or streptavidin.

Optionally, resistance compound includes saccharide part, polysaccharide, albumen, glycoprotein or polypeptide.

Optionally, resistance compound includes BSA, lysozyme, beta-actin, myosin, lactalbumin, egg white egg White, beta galactosidase, lactic dehydrogenase or immunoglobulin (such as IgG).

Optionally, reaction mixture also includes auxilin.

Optionally, auxilin includes unwindase, single strand binding protein or recombinase load factor.

Optionally, unwindase includes the uvsW from T4 bacteriophage.

Optionally, single strand binding protein includes the Sso for carrying out bin cure mine sulfolobus solfataricus (Sulfolobus solfataricus) SSB, the MjA SSB or Escherichia coli SSB albumen for coming from Methanococcus jannaschii (Methanococcus jannaschii).

Optionally, single strand binding protein includes gp32 albumen from T4 bacteriophage or from the modified of T4 bacteriophage Gp32 albumen.

Optionally, it includes the uvsY from T4 bacteriophage that recombinase, which is loaded into albumen,.

Optionally, reaction mixture also includes ATP.

Optionally, reaction mixture also includes ATP regenerating system.

Optionally, ATP regenerating system includes phosphocreatine.

Optionally, ATP regenerating system includes creatine kinase.

Optionally, reaction mixture also includes the adduct of the efficiency or yield for increasing nucleic acid amplification reaction.

Optionally, adduct include glycine betaine, DMSO, proline, trehalose, MMNO (4- methylmorpholine N-oxide) or PEG sample compound.

Optionally, at least one polynucleotide template in reaction mixture includes at least certain a part with the first primer Complementary or identical First ray, and at least a certain partial complementarity of the second primer or identical second sequence.Optionally, instead Answering mixture includes multiple double-stranded polynucleotides, and the polynucleotides include at least a certain partial complementarity or phase with the first primer With First ray, and at least a certain partial complementarity of the second primer or identical second sequence.Optionally, First ray position In multiple at or near the end of at least one double-stranded polynucleotide, and the second sequence is located at least one double-strand in multiple At or near another end of polynucleotides.

Optionally, reaction mixture also includes diffusion limitation agent.

Optionally, diffusion limitation agent reduces the diffusion rate that polynucleotides leave support.

Optionally, diffusion limitation agent reduces the level for being connected to the polyclonal nucleic acid group of support.

Optionally, diffusion limitation agent includes polymer compound.

Optionally, diffusion limitation agent includes glycopolymers.

Optionally, diffusion limitation agent includes the compound based on cellulose.

Optionally, diffusion limitation agent includes glucose or galactose polymer.

Optionally, glycopolymers include cellulose, glucan, starch, glycogen, agar or agarose.

Optionally, diffusion limitation agent includes block copolymer compound.

Optionally, diffusion limitation agent includes in poly- (propyIoxirane) flanked by two poly- (ethylene oxide) hydrophilic chains Heart chain.

Optionally, diffusion limitation dosage form is at micella.

Optionally, diffusion limitation dosage form is at micella liquid crystal.

Optionally, diffusion limitation agent includes Pluronics^TMCompound.

Optionally, it is about 0.025-0.8%w/v or about 0.05-0.7%w/v or about that reaction mixture, which further includes concentration, The diffusion of 0.075-0.6%w/v or about 0.1-0.5%w/v or about 0.2-0.4%w/v reduce agent.

It optionally, further include including for the composition of nucleic acid amplification and relevant system, device, kit and method Surface, matrix or the medium in multiple sites, wherein at least one site are operationally coupled to one or more sensors.

Optionally, multiple sites include reaction chamber, support, particle, particle, sphere, globule, filter, flow cell, hole, Ditch, tank therefor, gel or pipe inner wall.

Optionally, multiple sites can be arranged in random array or organized array.

Optionally, multiple sites can be in fluid communication with each other.

Optionally, at least one of multiple sites includes three dimensional chemical matrix.

Optionally, at least one of multiple sites can be covalently coupled to three dimensional chemical matrix.

Optionally, at least one of multiple sites includes acrilamide layer.Optionally, at least one of multiple sites includes It is covalently coupled to the nucleic acid of acrilamide layer.

Optionally, hydrophilic polymer matrix includes aquogel polymer matrix.

Optionally, polyacrylamide is conjugated in Oligonucleolide primers.

Optionally, hole has 0.1 micron to 2 microns of characteristic diameter.

Optionally, hole has 0.01 micron to 10 microns of depth.

In some embodiments, sensor includes field effect transistor (FET).FET may include ion-sensitive FET (ISFET), chemosensitivity field effect transistor (chemFET) or bioactivity field effect transistor (bioFET).

Optionally, one or more sensors are configured to detect the by-product that nucleotide is incorporated to.

Optionally, configurable one or more sensors detect chemical part in one or more the multiple sites In the presence of.

Optionally, one or more sensors include field effect transistor (FET), ion-sensitive field effect transistor (ISFET), chemosensitivity field effect transistor (chemFET) or bioactivity field effect transistor (bioFET).

In some embodiments, sensing material includes metal-oxide.

In some embodiments, sensing material is sensitive to hydrogen ion.

It optionally, include pyrophosphate, hydrogen ion or proton from the by-product that nucleotide is incorporated to reaction.

Additionally provide comprising either one or two of following, any subgroup or all compositions: at least one reversed nucleic acid chains is consolidated It is scheduled on multiple forward primers at least one support, multiple reverse primers and polymerase in solution.It is positive and/or reversed Primer is optionally low melting point or is rich in adenine, thymidine or uracil as described herein.Exemplary composition Including the solid support comprising multiple clone groups being spatially separated, each comfortable 3 ' end of clone group is drawn comprising low melting point Object binding sequence simultaneously includes low melting point primer sequence in 5 ' ends.Optionally, composition also includes recombinase.Alternatively, composition The enzyme that another kind is not polymerase, such as recombinase or reverse transcriptase or unwindase or nickase are not included optionally.It is another Exemplary composition includes any one or more of following component: (1) reversed nucleic acid chains, (2) fixed on the support multiple Low melting point forward primer, multiple low melting point reverse primers in (3) solution, and (4) polymerase.Optionally, forward primer and anti- It is interfertile (such as complementary) to 3 ' parts of chain or end.Optionally, the 5 ' parts or end of reverse primer and reverse strand It is substantially the same.Composition may include any one or more reagents described herein, and/or can undergo described herein Any one or more programs or condition.

In some embodiments, present disclosure is usually directed to the expansion comprising being generated by any method of present disclosure The composition of the nucleic acid of increasing.In some embodiments, clonal expansion is formed around discontinuous site on the support The clone group of limitation.Exemplary discontinuous site is tie point of the initial nucleic acid chain to support, and from the site using Beginning nucleic acid or its copy directly or indirectly generate other nucleic acid in clone group as template by primer extend.

Optionally, composition includes the gleanings for the nucleic acid that can be generated by any one or more methods described herein. For example, gleanings may include the nucleic acid for occupying the fixation in region of one or more separation on the surface.In some embodiments In, each region includes multiple identical nucleic acid chains and optionally multiple identical complementary strands for being hybridized to it, wherein complementary strand Do not adhere to solid support or connect or associate, in addition to because caused by hybridizing with fixed nucleic acid.Optionally, so fixed Individual nucleic acid chains in region as position are located on the surface within the distance of the length of the chain so as to another nucleic acid chains.Appoint Selection of land secures every mm on the surface of nucleic acid on it²On there are at least one separation region.For example, the number in isolated region Amount/mm²The surface for securing nucleic acid on it is greater than 10², be greater than 10³, be greater than 10⁴, be greater than 10⁵, be greater than 10⁶, be greater than 10⁷Or it is big In 10⁸。

The gleanings of the clone group of amplification can form array, can be that one-dimensional (such as a universal monoclonal of column is micro- Pearl) or two-dimensional (such as the clone group of amplification is located on plane support) or three-dimensional.Optionally but not necessarily positioning or The individual of arranged array clones group so that it is addressed or addressable.Optionally, different clones group is with appropriately distance It is spaced each other, the distance is typically enough to that different clone groups is allowed to be distinguished from each other.In embodiments, with orderly or unordered (such as random) mode spread on a planar substrate limitation clone group.

Exemplary array is characterized in the differentiable nucleic acid clone group of individual, wherein optionally in one or more supports Upper distribution this feature.In exemplary microballon embodiment, array includes multiple microballons, wherein individual microballon generally comprises nucleic acid Monoclonal group, different microballons generally comprises different clone groups (for example, it is different in sequence).Optionally, by microballon In the single layer of distribution or filling on a planar substrate.In other embodiments, array includes single (such as plane) support, Single support includes multiple spatially discontinuous nucleic acid clone groups, wherein different clone groups optionally in sequence not Together.

Optionally, one or more nucleic acid in individual clone group can be attached directly to planar substrate.In another reality In example, the nucleic acid of individual clone group is connected to microballon, such as discussed in this article.Optionally on a planar substrate with random Or orderly mode is tightly packed within microballon is cloned together.Optionally, more than 20%, 30%, 50%, 70%, 80%, 90%, 95% or 99% microballon at least one, two, the other microballons of four or six contact.Optionally, it is less than 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or 99% microballon and one, two, four or six it is other Microballon contact.

In some embodiments, present disclosure generally relates to the method and relevant combination of nucleic acid amplification Object, system, kit and device, including any amplification method, composition or system disclosed herein is used to carry out multi-kernel Acid amplification.

In some embodiments, method includes that multiplex amplification is carried out using polymerase is (such as polymerase-mediated multiple Nucleic acid amplification reaction).

In some embodiments, method, which may also include, is expanded using nucleic acid amplification reaction again from multiple nucleic acid amplification Amplicon.

Optionally, multiple nucleic acid amplification can carry out in single reaction mixture.

Optionally, multiple nucleic acid amplification can be carried out to the sample comprising multiple and different nucleic acid target sequences.

Optionally, multiple and different nucleic acid target sequences can be expanded in single reaction mixture.

Optionally, can be expanded in single reaction mixture it is a small number of beat it is at least hundreds of or at least it is thousands of (or more) Nucleic acid target sequence.

Optionally, at least 50 or at least 100 nucleic acid target sequences can be expanded in single reaction mixture.

Optionally, multiplex amplification may include by least partly sample and recombinase, polymerase and/or at least one primer Any or any combination is contacted.

Optionally, multiplex amplification can be carried out under isothermal or thermal cycle conditions.

In some embodiments, present disclosure generally relates to the method and relevant combination of nucleic acid amplification Object, system, kit and device, including multiple nucleic acid amplification, the multiple nucleic acid amplification includes in single reaction mixture From the sample comprising multiple and different nucleic acid target sequences expand at least 50 different nucleic acid target sequences (or more), the expansion Increase includes at least partly sample contacting under the conditions of isothermal duplication with recombinase and multiple primers.

In some embodiments, present disclosure generally relates to the method and relevant combination of nucleic acid amplification Object, system, kit and device, including multiple nucleic acid amplification, the multiple nucleic acid amplification includes in single reaction mixture Expand different nucleic acid target sequences from the sample comprising multiple and different nucleic acid target sequences, the amplification includes by will at least portion Point sample is contacted to generate at least 50 multiple and different under the conditions of isothermal duplication with recombinase and multiple primers Amplification target sequence (or more).

In some embodiments, present disclosure generally relates to the method and relevant combination of nucleic acid amplification Object, system, kit and device, including expanded by using nucleic acid amplification reaction (such as recombinase) and expanded from multiple nucleic acid again The amplicon of increasing generates the nucleic acid group of substantially monoclonal.

Optionally, the method for multiple nucleic acid amplification may also include the nucleic acid amplification method of recombinase-mediated comprising Expand at least some of the target sequence of at least 50 different amplifications again as follows: it includes single continuous for (a) being formed The reaction mixture of liquid phase, it includes (i) multiple supports, the target sequence of (ii) 50 different amplifications at least one and (iii) recombinase；(b) reaction mixture is made to undergo amplification condition, to generate multiple supports, the multiple support connects It is connected to the nucleic acid group for being connected to it of substantially monoclonal.

Optionally, in the method expanded for multiple nucleic acid, can substantially it is non-exhaust under conditions of amplification from sample This different nucleic acid target sequences.

Optionally, in the method expanded for multiple nucleic acid, it can be expanded under conditions of substantially used up and come from sample Different nucleic acid target sequences.

Optionally, in the method expanded for multiple nucleic acid, single reaction mixture includes isothermal or thermal cycle reaction Mixture.

In some embodiments, two or more templates can be expanded in the isolated room of array, hole, chamber or site Or target, isolated room, hole, chamber or the site of the array are in fluid communication with each other or are amplified the identical single of reaction mixture Continuous Liquid Phase occupies.Such embodiment includes the embodiment for the nucleic acid amplification based on array.

For example, in some embodiments, this disclosure relates to the methods for nucleic acid amplification, comprising: by target multicore Thuja acid distributes reaction chamber or site into reaction chamber or the array in site, and single target multicore is expanded in reaction chamber or site Thuja acid.Optionally, two or more target polynucleotides are distributed into two or more reaction chambers of array or site, and And two or more the allocated target polynucleotides are amplified in parallel in its respective reaction chamber or site.Optionally, At least two of reaction chamber or site respectively receive single target polynucleotide (one or more of reaction chamber or site during distribution It is a optionally to receive zero or more than one target polynucleotide during distribution).At least two target polynucleotides can be each at it From reaction chamber in expand with being cloned.At least one reaction chamber comprising target polynucleotide can with comprising target polynucleotide extremely Few other reaction chambers are in fluid communication during amplification.

In some embodiments, present disclosure generally relates to method (and the relevant combination of nucleic acid amplification Object, system, device and kit), comprising: (a) is anti-by the way that single polynucleotides introducing at least two is in fluid communication with each other Room is answered to distribute at least two different polynucleotides into the array of reaction chamber；(b) by anti-described at least two Answer the nucleic acid group of indoor amplifying polynucleotides formation at least two substantially monoclonal.Normally, at least two reaction chambers are expanding It keeps being in fluid communication each other during increasing.

In some embodiments, present disclosure generally relates to method (and the relevant combination of nucleic acid amplification Object, system, device and kit), comprising: (a) include the first and second reaction chambers reaction chamber array in, by the first mould Plate polynucleotides distribute into the first reaction chamber and distribute the second template polynucleotide into the second reaction chamber, and (b) pass through Clone the first and second template polynucleotides are expanded in its respective reaction chamber to form at least two substantially monoclonals Nucleic acid group, wherein distributing single polynucleotides from the sample of nucleic acid with multiple and different polynucleotides.Optionally, the first He Second reaction chamber includes the different piece of single Continuous Liquid Phase during amplification.For example, the first and second reaction chambers of array can It is in fluid communication during amplification.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, including (a) will be different Single polynucleotides are distributed into each of multiple reaction chambers, and (b) different single by expanding in multiple reaction chambers Polynucleotides to form monoclonal nucleic acid group in each of reaction chamber, wherein from the core with multiple and different polynucleotides The single different polynucleotides of acid sample distribution.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification is included (a) by will be single A polynucleotides introduce the reaction chamber that at least two are in fluid communication with each other and distribute at least two different polynucleotides to anti- It answers in the array of room；(b) by least two reaction chamber, to form at least two substantially single for amplifying polynucleotides The nucleic acid group of clone.

In some embodiments, method, which may also include, introduces one or more supports (such as globule or particle etc.) At least one reaction chamber of array or site.Can before, during or after distributing polynucleotides into array by one or Multiple supports introduce at least one reaction chamber or site.In some embodiments, at least one reaction chamber of array or position Point receives single support.In some embodiments, most of reaction chamber or site receive single support.In some implementations In scheme, support can be mixed before a distribution with polynucleotides and distribute it into array together with polynucleotides.Extremely A few support is optionally connected to nucleic acid molecules, the nucleic acid molecules include be present in during amplification reaction chamber or The part of polynucleotides in site is substantially complementary or substantially the same primer sequence.In some embodiments, at least One support includes nucleic acid molecules, and the nucleic acid molecules include the part with target polynucleotide or template in reaction chamber or hole It is substantially complementary or substantially the same primer sequence.In some embodiments, at least one support includes nucleic acid molecules, The nucleic acid molecules include with another primer that is present in reaction chamber or site during amplification at least partly substantially Complementary or substantially the same primer sequence.

In some embodiments, amplification may include at least one reaction chamber or the site that reaction mixture is introduced to array In.Optionally, reaction is mixed before, during or after the introducing in the distribution or support of polynucleotides to array to array Object is introduced into reaction chamber or site.It can in any order or any combination draws reaction mixture, support and polynucleotides Enter or distributes into array.In some embodiments, at least one reaction chamber of array or site receive single support, list A polynucleotides and reaction mixture enough to support amplification of the polynucleotides in reaction chamber or site.

In some embodiments, method may include being incited somebody to action by contacting support under hybridization conditions with polynucleotides Polynucleotides are at least partly hybridized to support.Hybridization can be in the reaction chamber of support and/or polynucleotides to array or position Occur before, during or after the introducing of point.In some embodiments, at least one branch of the first primer sequence will be connected to Hold at least one reaction chamber or site that object introduces array, polynucleotides then introduced into reaction chamber or site, in reaction chamber or Polynucleotides are hybridized to support in site.Alternatively, support can be hybridized to through the polynucleotides in reaction chamber or site Expand the amplified production generated.

Reaction mixture may include any reaction mixture described herein and component.In some embodiments, instead Answering mixture may include any one or more of following component: isothermal duplication reagent is (for example, one or more recombinases, untwist Enzyme, that is, relevant confactor, polymerase etc.), screening agent, nucleotide etc..

In some embodiments, present disclosure generally relate to nucleic acid amplification method (and relevant composition, Kit, system and device), comprising: (a) is in any order or combination introduces the first polynucleotide template and the first support The first reaction chamber or site of reaction chamber or the array in site, and the second polynucleotide template and the second support are introduced into array The second reaction chamber or site；(b) clone the first multicore is expanded on the first support in the first reaction chamber or site Thuja acid template, and the second polynucleotide template is expanded to clone on the second support in the second reaction chamber or site, simultaneously The first reaction chamber or site and the second reaction chamber or site are in fluid communication during amplification.The amplification of clone ground may include generating connection Extremely the first support of the first amplicon from the first polynucleotide template, and be connected to from the second polynucleotide template Second support of the second amplicon.Optionally, the first and second sites (or reaction chamber) includes same reaction during amplification The identical Continuous Liquid Phase of mixture.Such as reaction mixture may include comprising the first and second polynucleotide templates and first and the The single Continuous Liquid Phase of two supports.It can will be reacted before, during or after the introducing of polynucleotide template and/or support Mixture introduces reaction chamber or the site of array.In some embodiments, disclosed method further includes introducing first and the Reaction mixture is introduced into the first and second reaction chambers or site after two polynucleotide templates and the first and second supports.? In some embodiments, reaction mixture includes recombinase or unwindase or recombinase and unwindase.Recombinase can derive from flesh Tail virus (such as uvsX), bacterium, yeast or people recombinase or its analog from other species.In some embodiments In, reaction mixture includes polymerase.In some embodiments, reaction mixture include screening agent, such as polyacrylamide, Agarose or cellulosic polymer (for example, HEC, CMC or MC or derivatives thereof).In some embodiments, reaction mixture Agent is limited comprising diffusion.

In some embodiments, amplification includes that the first primer binding sequence of polynucleotide template is hybridized to support The first primer the first primer sequence by the polynucleotide template be connected to comprising the first primer sequence first The support of primer or surface (such as particle or globule).

In the embodiment wherein expanded in the reaction chamber of array or site and wherein in single reaction vessel In the embodiment inside expanded, surface or support optionally include at least the first primer, and it includes the first primer sequences. In some embodiments, one or more polynucleotide templates include the first primer binding sequence.The first primer binding sequence It can be identical or substantially the same with the first primer sequence.Alternatively, the first primer binding sequence can be with the first primer sequence It is complementary or is substantially complementary.In some embodiments, the first primer sequence and the first primer binding sequence are not shown Significant identity or complementarity, but it is substantially the same or basic with another kind nucleotide sequence present in reaction mixture Upper complementation.In such embodiments, amplification may include the formation of amplified reaction intermediate product, the intermediate product include with The first primer sequence, the first primer binding sequence or both have the nucleotide sequence of significant identity or complementarity.

In some embodiments, there are at least two different polynucleotide templates in the reactive mixture, and expand Increase the formation for leading at least two different substantially monoclonal groups, the monoclonal group respectively derives from the single multicore glycosides The amplification of acid template.In some embodiments, at least two substantially monoclonal groups two or more be connected to it is identical Support or surface.Each of two or more substantially monoclonal groups can be connected on identical support or surface Different unique locations.Alternatively, each of two or more substantially monoclonal groups can be each attached to different branch Hold object or surface.The separation on different monoclonal group to different supports or surface is needing segregating population before analysis It can be advantageous in.In some embodiments, support or surface are the parts of globule or particle, in shape It can be spherical or sphere.In some embodiments, support or surface form the part of two dimension or cubical array.

In some embodiments, disclosure is carried out when agent being reduced comprising screening agent or diffusion in the reactive mixture The method for nucleic acid amplification.These reagents can increase the total quantity and/or ratio of the monoclonal group formed during amplification (such as percentage).In some embodiments, method includes use relative to the increased Dan Ke of popular response mixture offer The reaction mixture of the yield of grand or substantially monoclonal amplicon.

In some embodiments, method includes being expanded by partial denaturation template.For example, amplification includes template row It walks.For example, template to be amplified may include the joint sequence comprising primer binding site, integrally there is phase compared to template To low Tm.In some embodiments, as being more fully described herein, in the Tm for being substantially higher than joint sequence and base In sheet lower than template Tm at a temperature of expanded.In some embodiments, lower than at least 5 DEG C of nucleic acid-templated Tm, It is expanded at a temperature of 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C.In some embodiments, it is being higher than (for example, at least 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C or 50 DEG C higher) the first primer, the second primer or the first and second primers Tm temperature Under expanded.

In some embodiments, reaction mixture optionally includes any one or more of following component: (a) optionally Ground includes one or more supports of at least the first primer sequence；(2) recombinase；(3) polymerase；(4) diffusion limitation agent； (5) agent is sieved；(6) crowding agent；(7) ATP regenerating system；(8) single strand binding protein (SSBP)；(8) recombinase auxiliary because Son, such as recombinase are loaded into albumen.In some embodiments, by diffusion limitation agent and/or screening agent there are the case where Under on the surface amplification (for example, by the way that an amplimer to be connected on surface) polynucleotide template come increase monoclonal or The substantially yield of the group of monoclonal.The product multicore glycosides that agent can be expanded during amplification by reduction is sieved in diffusion limitation agent Acid leaves diffusion or the migration on surface to provide the yield of increased monoclonal group.

In some embodiments, reaction mixture includes one or more isothermal duplication reagents.Such reagent can wrap Include such as recombinase or unwindase.

In some embodiments, such as compound to be formed in combination with the first primer using the enzyme of catalysis homologous recombination Object can be catalyzed chain intrusion or can form the enzyme of D- ring structure, to carry out the method for nucleic acid amplification.In some embodiments In, the enzyme for being catalyzed homologous recombination includes recombinase.

In some embodiments, amplification condition includes isothermy or thermal cycle conditions.

It in some embodiments, include: that (a) forms the reaction including single Continuous Liquid Phase for the method for nucleic acid amplification Mixture, the single Continuous Liquid Phase include the enzyme of (i) catalysis homologous recombination, (ii) one or more surfaces, and (iii) multiple Different polynucleotides；(b) reaction mixture experience is made to be suitable for the condition of nucleic acid amplification.

It in some embodiments, include: that (a) forms the reaction including single Continuous Liquid Phase for the method for nucleic acid amplification Mixture, the single Continuous Liquid Phase include the enzyme of (i) catalysis homologous recombination, and (ii) one or more is each attached to multiple the The globule of one primer, and the polynucleotides that (iii) is multiple and different；(b) by making reaction mixture that amplification condition be undergone to form two The nucleic acid group of the amplification of a or more substantially monoclonal.Amplification condition may include isothermal or thermal cycle conditions.In some realities It applies in scheme, the first primer can be hybridized to polynucleotides at least partly.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, comprising: (a) forms packet Include the reaction mixture of single Continuous Liquid Phase, the single Continuous Liquid Phase includes one or more supports (or surface), multiple Polynucleotides and recombinase；(b) by making reaction mixture undergo amplification condition at least one support (or surface) on gram At least two of the plurality of different polynucleotides are expanded grandly.In some embodiments, amplification condition may include isothermal Or thermal cycling amplification condition.Reaction mixture optionally includes recombinase.In some embodiments, reaction mixture includes Polymerase.In some embodiments, reaction mixture includes primer, and the primer can be in the solution.Optionally, support Or at least one of surface may include primer.

In some embodiments, the method that present disclosure generally relates to nucleic acid amplification, comprising: (a) forms packet The reaction mixture of single Continuous Liquid Phase is included, the single Continuous Liquid Phase includes (i) recombinase, and (ii) is connected to one or more Multiple globules of the first primer comprising the first primer sequence, and the polynucleotide template that (iii) is multiple and different；It (b) will be described At least one of the first primer is hybridized at least one of multiple and different polynucleotide templates；(c) undergo reaction mixture Nucleic acid amplification condition and at least one is generated to form at least first amplification group by expanding at least one polynucleotide template The substantially polynucleotides group of monoclonal.In some embodiments, at least one substantially in the group of monoclonal at least 30%, 90% polynucleotides and original at least one polynucleotide template being present in reaction mixture are substantially the same (or being substantially complementary).In some embodiments, one for being at least partly connected to multiple globules of the first amplification group is small Pearl.

In some embodiments, the formation reaction mixture in step (a) include: by by recombinase and be connected to it is more At least one of multiple the first primers of a globule is contacted to form nucleoprotein complex.

In some embodiments, reaction mixture experience nucleic acid amplification condition is made to include carrying out nucleosides in step (b) Sour polymerization reaction.For example, nucleotide polymerization reaction may include optionally when the first primer sequence is hybridized in reaction mixture Nucleotide is incorporated to the first primer sequence when one polynucleotide template.

In some embodiments, making reaction mixture experience nucleic acid amplification condition includes by the first primer and polynucleotides Template, recombinase, polymerase and nucleotide are contacted in any order or with any combination.

In some embodiments, nucleic acid amplification condition includes repeating such circulation: being formed and is drawn comprising recombinase, first Object at least partly at least part of nucleoprotein complex of the first polynucleotide template, and by nucleoprotein complex and catalysis The polymerase of one or more nucleotide to the first primer being incorporated to is contacted.

In some embodiments, the nucleic acid amplification reaction that can be recycled respectively is connected to substantially monoclonal to generate Polynucleotides group multiple globules.

In some embodiments, the method for nucleic acid amplification can be carried out under isothermy or thermal cycle conditions.

In some embodiments, multiple and different polynucleotides can be single-stranded or double-stranded polynucleotides.In some realities It applies in scheme, the thermal denaturation or chemical modification of double-stranded polynucleotide are non-required, because recombinase can pass through catalysis chain intrusion Generate the chain denaturation of part.

In some embodiments, the method for nucleic acid amplification can be carried out in single reaction vessel.In some implementations In scheme, nucleic acid amplification reaction can be carried out in the single reaction vessel comprising single Continuous Liquid Phase.For example, single Continuous Liquid Phase It may include amplification mixture, the amplification mixture includes multiple globules for being respectively connected to multiple the first primers, multiple and different Polynucleotides and multiple recombinases.In some embodiments, amplification mixture may also include polymerase and multiple nucleotide. In some embodiments, amplification mixture may also include ATP, nucleotide and confactor.Individually reaction vessel is unrestricted Property example includes pipe, hole or similar structure.

It in some embodiments, can in any order include that combination continuously or substantially simultaneously or both will Polynucleotides and reagent are placed into reaction vessel.In some embodiments, reagent includes being connected to multiple the first primers Globule, recombinase, polymerase, nucleotide, ATP, bivalent cation and confactor.

In some embodiments, the method for nucleic acid amplification can be carried out in single Continuous Liquid Phase.Single continuous liquid It mutually may include any given part or any other portion in region and identical single Continuous Liquid Phase of wherein single Continuous Liquid Phase Point or regional fluid connection any liquid phase.Normally, the component being dissolved or suspended in single Continuous Liquid Phase can freely expand Dissipate or migrate any other point into liquid phase.In some embodiments, however, single Continuous Liquid Phase may include diffusion limitation Agent slows down the diffusion rate in single Continuous Liquid Phase.One exemplary implementation scheme of single Continuous Liquid Phase is water-in-oil type Single aqueous droplet in emulsion；In such emulsion, each droplet will form isolated phase；Two combinable shapes of droplet At single phase.

In some embodiments, single Continuous Liquid Phase is substantially made of single water phase.In some embodiments, single One Continuous Liquid Phase lacks nonaqueous phase；For example, Continuous Liquid Phase does not include oil or organic solvent.In some embodiments, Duo Gehe Sour amplified reaction occurs in the water phase of single reaction vessel.In some embodiments, single Continuous Liquid Phase is not divided in list Multiple nucleic acid amplification reactions occur in a reaction vessel.

In some embodiments, the method for nucleic acid amplification can be carried out in the water-in-oil emulsion divided is provided.

When using multiple polynucleotide templates carry out nucleic acid amplification when, using conventional amplification method clonal expansion usually according to Rely the isolated part or component that reaction mixture is for example divided to the connection of nonfluid each other in technology, with keep it is Clonal and It prevents the cross contamination of different amplification groups and keeps the yield of enough monoclonal amplified productions.Use such conventional expansion Increasing method, clone's ground amplifying polynucleotides template in identical reaction mixture and without reaction mixture to isolated area The division or distribution of room or container are usually infeasible, because of any multicore in such amplification present invention mixture Thuja acid (including template and/or amplified production) will tend to randomly migrate by mixture because of diffusion and/or Brownian movement. Such diffusion or migration usually increase the generation of polyclonal amplification, so that few (if any) monoclonal will be generated Group.

Physical barriers are used to reduce a technology appropriate of the generation of polyclonal group in conventional amplification method Individual amplified reaction is separated into discontinuous compartment.For example, emulsion PCR uses the micro- reaction vessel of water-in-oil type, wherein oil It mutually include the i.e. discontinuous aqueous reaction compartment of many separation.Each compartment is used as independent amplified reaction container, thus Entire emulsion can be supported to be permitted in separation (discontinuous) liquid phase in single reaction vessel (such as Eppendorf pipe or hole) The amplified reaction more separated.Similarly, amplification " Master Mix " can be prepared and distributed to isolated reaction chamber (such as hole Array) in, generate one group of discontinuous and isolated phase, the isolated amplified reaction of each of which restriction.It can be incited somebody to action before amplification Such separation is mutually further closed each other.Such closing can be used for preventing the intersection between parallel and isolated reaction dirty Dye.Closed exemplary form may include using lid or phase barrier (such as mineral oil layer on aqueous reaction) to incite somebody to action PCR reaction is divided to individual and discontinuous compartment, and the transfer of reactive component will not occur between the compartment.

Depend on the one or more during amplification anti-to prevent cross contamination and reduce other technologies of polyclonal property Answer the cross contamination for being fixed against amplified reaction product of component (such as one or more templates and/or primer) causes with it Monoclonicity reduction.One such example includes bridge-type PCR, wherein all primers required for amplification (such as it is positive And reverse primer) it is connected to the surface of matrix support.It in the reactive mixture may include other in addition to such fixation Fixed component.For example, polynucleotide template and/or amplimer can be suspended in during amplification in gel or other matrix with Prevent amplified reaction product from migrating from synthesis site.Such gel and matrix usually require to be removed later, this needs makes With " unwinding " appropriate or other recycling step, to lose yield.

In some embodiments, present disclosure provide in the single Continuous Liquid Phase of reaction mixture in parallel The amplification for carrying out the substantially monoclonal of multiple polynucleotide templates exists without multiple reactive components (such as two kinds of primers) The method of division or fixation during amplification.It alternatively, can be directly by the mixture of the polynucleotide template in solution and expansion The surface appropriate or support for increasing reactive component and the first primer attached thereto are contacted, can be identical continuous Other components needed for providing amplification in liquid phase, draw including polymerase, the nucleotide of one or more types and optionally second Object.In some embodiments, reaction mixture further includes recombinase.Optionally, reaction mixture further includes at least one choosing The reagent that self-diffusion limits agent, sieves agent and crowding agent.It include to be suitable for carrying out template in single Continuous Liquid Phase The example of the amplification mixture of monoclonal amplification is further described through in this article.It optionally, can be in single surface or support On different positions on expand different templates, can in identical reaction mixture different surfaces or different branch It holds and expands different templates on object.

In some embodiments, reaction mixture may include one or more screening agent.Screening agent optionally includes can Any compound of physical barriers is provided to the migration of polynucleotide template or its corresponding amplified production.(migration may include mould Any movement of plate or amplified production in reaction mixture；Diffusion includes involving the migration form moved along concentration gradient).? In some embodiments, screening agent includes that can provide any compound of the matrix with multiple apertures, the multiple aperture foot Enough small movements to reduce any one or more specific components of nucleic acid synthesis reaction mixture or nucleic acid reaction mixture.

In some embodiments, screening agent provides molecular sieve.For example, screening agent can reduce polynucleotides (or and surface Or the polynucleotides of globule association) by the inclusion of the movement for the reaction mixture for sieving agent.Screening agent optionally has aperture.

When in the single Continuous Liquid Phase in reaction mixture clone expand two or more template polynucleotides when It waits, sieve agent includes that can be advantageous.For example, screening agent can prevent or slow down template or at least a certain portion by template Point diffusion of the polynucleotides in reaction mixture of amplification that generates of duplication, thus prevent the formation of polyclonal pollutant and Reaction mixture is divided without passing through physical method or packaging method (such as emulsion) during amplification.It is such individually to react Clone ground amplification template is considerably reduced and is generated without the method divided and is suitable in the single Continuous Liquid Phase of mixture It shares in the relevant cost in library, time and the workload of high throughput method such as digital pcr, next-generation sequencing etc..

In some embodiments, the average pore size for sieving agent is so that target component (such as polynucleotides) is mixed in reaction Movement in object is hindered or is prevented by selectivity.In an example, screening agent includes that can provide the matrix with multiple apertures Any compound, the multiple aperture it is sufficiently small with slow down or hinder polynucleotides by the inclusion of screening agent reaction mixture Movement.Therefore, screening agent can reduce the Brownian movement of polynucleotides.

In some embodiments, screening agent selectively acting is to hinder with the average mark higher than certain threshold value or range The migration of the molecule of sub- size or weight, without hindering other mean molecule sizes or weight having lower than threshold value or range Molecule migration.

In some embodiments, screening agent selectively acting is to hinder with the average mark lower than certain threshold value or range The migration of the molecule of sub- size or weight, without hindering other mean molecule sizes or weight having higher than threshold value or range Molecule migration.

In some embodiments, screening agent may be selected selectively to hinder, slow down, reduce or polynucleotides is prevented to pass through The movement of reaction mixture, but it is sufficiently large to allow lesser component (for example, cation, nucleotide, ATP and confactor) logical Cross the movement of reaction mixture.In some embodiments, screening agent have can by increased or decrease screening agent concentration come The average pore size or average pore size scope of adjustment.For example, the molecular weight of screening agent (or combination of screening agent), intrinsic viscosity may be selected Spend with concentration and prepare nucleic acid reaction mixture in specific solvent (such as water) to generate such matrix: it is with desired Prevent the ability with the migration of the target polynucleotide of particular size or length or desired average pore size or viscosity.Some In embodiment, screening agent can reduce bulk flow by increasing the viscosity of nucleic acid reaction mixture.In some embodiments In, screening agent can be water-soluble.It in some embodiments, can be by the way that agent and solvent (such as aqueous solvent, example will be sieved Such as water) it mixes to prepare the matrix with multiple apertures.In some embodiments, screening agent does not interfere recombinase nucleoprotein multiple Close formation or the nucleotide polymerization of object.

In some embodiments, present disclosure generally relates to the method for carrying out nucleic acid amplification reaction, including logical Cross there are one or more screening agent, optionally there are recombinase, polymerase or any other it is appropriate can be catalyzed or Promote to expand target polynucleotide on surface or support in the case where the reagent of nucleic acid amplification to generate two or more bases The group of monoclonal in sheet.

In some embodiments, polynucleotides can be reduced including screening agent in the reactive mixture and leaves given support Or surface movement (such as reduce fall off) and polynucleotides can be increased will be hybridized to support or surface and nucleotide is provided gather A possibility that starting position of conjunction, to increase the ratio of the amplicon of the substantially monoclonal generated during amplified reaction.

In some embodiments, amplification include exist sieve agent in the case where on multiple and different globule supports Multiple and different polynucleotide templates is expanded, and recycles the globule support of the substantially monoclonal of certain percentage, each The globule support of such substantially monoclonal includes the globule support for being connected to the polynucleotides group of substantially monoclonal. In some embodiments, the percentage of the globule support of the substantially monoclonal of recycling is substantially higher than from reaction mixture The globule support (include polyclonal or monoclonal group total globule support) of total amplification of recycling 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 90% or 95%. In some embodiments, the percentage of the globule support of the substantially monoclonal of recycling, which is substantially higher than, is being not present screening It is recycled after being expanded under in the case where agent but in other aspects essentially similar or identical amplification condition substantially single The percentage of the globule support of clone.

In some embodiments, screening agent includes polymer compound.In some embodiments, screening agent includes handing over Connection or noncrosslinking polymer compound.By way of non-limiting example, screening agent may include polysaccharide, polypeptide, You Jiju Close object etc..

In some embodiments, screening agent includes linear or branch polymer.In some embodiments, agent is sieved Including with point or neutral polymer.

In some embodiments, screening agent may include one or more respectively with the polymerization of average molecular weight and viscosity The mixture of object.

In some embodiments, screening agent include have about 10,000-2,000,000 or about 12,000-95,000 or The polymer of the average molecular weight of about 13,000-95,000.

In some embodiments, screening agent can be displayed in when being dissolved in the water with 2 weight percent at about 25 DEG C About 5 centipoises of measurement are to about 15,000 centipoises, or about 10 centipoises measured at about 25 DEG C with 2% aqueous solution are to about 10,000 lis It moors, or the average viscosity range of about 15 centipoises to about 5,000 centipoises measured at about 25 DEG C with 2% aqueous solution.

In some embodiments, screening agent includes about 25 to about 1,5000kM_vOr about 75-1,000kM_vOr about 85- 800kM_vViscosity-average molecular weight (M_v).In some embodiments, reaction mixture includes about 0.1 to about 20% weight/body Long-pending or about 1-10%w/v or about 2-5%w/v screening agent.

In some embodiments, screening agent includes acrylamide polymer such as polyacrylamide.

In some embodiments, screening agent includes the polymer of one or more amino acid.For example, screening agent may include Polylysine, polyglutamic acid, actin, myosin, keratin, tropomyosin (tropmyosin) etc..In some realities It applies in scheme, screening agent may include the derivative of any polypeptide of these polypeptides.

In some embodiments, screening agent includes polysaccharide polymer.In some embodiments, screening agent includes grape The polymer of sugar or galactolipin.In some embodiments, screening agent includes one or more selected from cellulose, glucan, shallow lake Powder, glycogen, agar, chitin, pectin or agarose polymer.In some embodiments, screening agent includes glucopyra Glycopolymers.

In some embodiments, it is polarity or electrification under amplification reaction condition that screening agent, which includes with one or more, Group polymer.For example, polymer may include one or more cation groups, one or more anionic groups or two Person.In some embodiments, screening agent is the polysaccharide comprising one or more charged groups.In some embodiments, it sieves Dividing agent is the polysaccharide comprising one or more carboxyls, and the carboxyl is or is intended to electronegative under amplification reaction condition. Such as screening agent may include carboxymethyl cellulose (CMC) polymer.In some embodiments, screening agent may include spermine and/ Or spermidine.In some embodiments, screening agent includes polylysine and/or poly arginine.For example, screening agent may include poly- L-lysine, poly- D-Lys, poly- D-Glu etc..In some embodiments, screening agent includes one or more group eggs White or histone-nucleic acid complexes or derivatives thereof.Histone is high alka albumen, can be in conjunction with nucleic acid and including albumen H1, H2A, H2B, H3 and H4.In some embodiments, such as pass through methylation, acetylation, phosphorylation, ubiquitination, SUMO Histone is modified in change, citrullinated, ribosylation (including ADP- ribosylation) etc..

In some embodiments, screening agent includes polymer, the polymer including chemical substitute.Polymer may include anti- Answer group (for example, reactive group can be reacted with substituent appropriate to generate substituted polymer).In some embodiments In, polymer includes fluorescence-, carboxyl-, amino-or the substituted polymer of alkoxy-.In some embodiments, pass through methyl Change, acetylation, phosphorylation, ubiquitination, carboxylated etc. carry out modified polymer.Substituent may include charged group, such as anion Or cation group, substitution of the group into polymer chain can lead to the generation of electropolymer.Substituted degree can be about 0.2 between about 1.0 derivatives/monomeric unit, usually between about 0.4 to about 1.0, more generally about 0.6 to about 0.95 Between change.

In some embodiments, screening agent includes cellulose derivative, such as sodium carboxymethylcellulose, carboxymethyl 2- hydroxyl Ethyl cellulose sodium, methylcellulose, hydroxyethyl cellulose, 2- hydroxypropyl cellulose, carboxymethyl cellulose, hydroxy propyl cellulose Element, hydroxyethylmethylcellulose, hydroxy butyl methyl cellulose, (hydroxypropyl) methylcellulose or hydroxyethyl ethylcellulose or packet Any one or more mixtures containing such polymer.

In some embodiments, nucleic acid reaction mixture includes the mixture of different screening agent, such as different fibres Tie up the mixture of plain derivative, starch, polyacrylamide etc..

In some embodiments, screening agent includes one or more complexing polymers, the complexing polymer packet Subdivision containing the different polymer of any two (including any polymer described herein).For example, complexing polymer It may include the polysaccharide for being connected to the polysaccharide polymer of polynucleotides polymer, such as being connected to DNA or RNA.In some embodiment party In case, screening agent may include the polymer comprising cellulosic sections and nucleic acid moiety, such as DNA- cellulose.In other embodiment party In case, complexing polymer may include the polyacrylamide for being connected to polynucleotides and/or polypeptide.Include in the reactive mixture Such complexing polymer can be used for further hindering movement of the target polynucleotide by reaction mixture.

In some embodiments, screening agent may include first with cross-linking agent appropriate or the polymer reacted. For example, screening agent may include the acrylamide reacted with bisacrylamide and/or two (acryloyl) cystamines.

In some embodiments, reaction mixture includes that at least one diffusion reduces agent.In some embodiments, expand Dissipate reduce agent include reduce polynucleotides from higher concentration region to the region with low concentration migration any compound. In some embodiments, diffusion reduces appointing for the migration that agent includes any component (no matter large or small) for reducing nucleic acid amplification reaction What compound.In some embodiments, the component of nucleic acid amplification reaction includes globule/primer, polynucleotides, recombinase, gathers Synthase, nucleotide, ATP and/or confactor.

It should be noted that the concept that screening agent and diffusion reduce agent is not certain mutually exclusive；Screening agent can usually have Effect ground reduces diffusion of the target compound by reaction mixture, and the effect of the screening to reactive component can usually be had by spreading reduction agent It answers.In some embodiments, identical compound or reaction mixture additive can be used as sieving agent and/or diffusion is reduced Agent.Any screening agent disclosed herein can be linked up in some embodiments is used as diffusion reduction agent, and vice versa.

In some embodiments, it includes polyacrylamide, agar, agarose or fibre that diffusion, which reduces agent and/or screening agent, Tie up plain polymer such as hydroxyethyl cellulose (HEC), methylcellulose (MC) or carboxymethyl cellulose (CMC).

In some embodiments, sieve agent and/or diffusion reduce agent at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 74%, 90% or 95%w/v (weight/reaction mixture unit volume of reagent) Concentration include in the reactive mixture.

In some embodiments, reaction mixture includes at least one crowding agent.For example, crowding agent can pass through production Raw crowded reaction environment increases the concentration of one or more components in nucleic acid amplification reaction.In some embodiments, it reacts Mixture includes screening agent and crowding agent.

It in some embodiments, include one or more surfaces for the method for nucleic acid amplification.In some embodiments In, surface can be connected with multiple the first primers, it is multiple in the first primer there is common the first primer sequence.

In some embodiments, surface can be the outside of object or uppermost layer or boundary.In some implementations In scheme, surface can be the inside on the boundary of object.

In some embodiments, reaction mixture includes multiple and different surface, such as reaction mixture may include more A globule (such as particle, nano particle, particle etc.) and at least two different polynucleotide templates can be on different surfaces On expand with being cloned, to form at least two different surfaces, each of the surface is connected to amplicon.Some In embodiment, reaction mixture includes single surface (for example, array of the surface of glass slide or reaction chamber) and at least two Two different regions or position are amplified a different polynucleotide template on the surface, are connected to two or more to be formed The single surface of multiple amplicons.

In some embodiments, surface can be porous, semi-porous or non-porous.In some embodiments In, surface can be plane surface and concave surface, convex surface or any combination thereof.In some embodiments, surface can be small Pearl, particle, particle, sphere, filter, flow cell, hole, ditch, tank therefor, gel or capillary inner wall.In some embodiments In, surface includes the inner wall of capillary, slot, hole, ditch, slot, container.In some embodiments, surface may include texture (example Such as, etching, coelosis, aperture, three-dimensional rack or bulge).

In some embodiments, particle can have sphere, hemispheroidal, cylindrical body, barrel-shaped, annular, bar Shape, plate-like, it is conical, triangle, cube shaped, polygon.Tubulose, linear or irregular shape Shape.

In some embodiments, surface can be prepared from any material, the material include glass, Pyrex, silica, Quartz, vitreous silica, mica, polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylates (PMA), poly- first Base methyl acrylate (PMMA), dimethyl silicone polymer (PDMS), silicon, germanium, graphite, ceramic, silicon, semiconductor, high refraction Rate dielectric, crystal, gel, polymer or film (for example, film of gold, silver, aluminium or diamond).

In some embodiments, film can be magnetic or paramagnetic beads (for example, magnetic or paramagnetic nanoparticle Or particle).In some embodiments, paramagnetic particles can be the paramagnetic beads (example for being connected to streptavidin Such as come from Invitrogen, the Dynabeads of Carlsbad, CA^TMM-270).Particle can have iron core or comprising hydrogel Or agarose (such as Sepharose^TM)。

In some embodiments, surface can be connected with multiple the first primers.Surface can be coated with acrylamide, carboxyl or Aminated compounds is to connect nucleic acid (such as the first primer).In some embodiments, amido modified nucleic acid (such as can be drawn Object) it is connected to the surface coated with carboxylic acid.In some embodiments, amido modified nucleic acid can be reacted with EDC (or EDAC) To be connected to the surface of carboxylic acid coating (with or without NHS).The acryloyl amination that the first primer can be fixed on surface Close object coating.It is biotinylated to combine that particle can be coated with avidin sample compound (such as streptavidin) Nucleic acid.

In some embodiments, surface includes the surface of globule.In some embodiments, globule includes polymeric material Material.For example, globule includes gel, hydrogel or acrylamide polymer.Globule can be porous.Particle can have chamber or small Hole, or may include three-dimensional rack.In some embodiments, particle can be Ion Sphere^TMParticle.

In some embodiments, disclosed method (and relevant composition, system and kit) include by one or It is multiple nucleic acid-templated to be fixed on one or more supports.It can include but is not limited to by any method physical absorption, pass through Ion or covalent bond formation or combinations thereof, by cDNA chip on solid support.Solid support may include polymerization, glass or Metal material.The example of solid support includes film, plane surface, microwell plate, globule, filter, test-strips, glass slide, lid glass Piece and test tube.Mean any solid phase material for synthesizing on it, adhering to, connecting or fix in other ways oligomer.Support Optionally including " resin ", " phase ", " surface " and " support ".Support can be by organic polymer such as polystyrene, poly- Ethylene, polypropylene, polyvinyl fluoride, polyethyleneoxy (polyethyleneoxy) and polyacrylamide and its copolymer and transplanting Object composition.Support can also be inorganic, such as glass, silica, controlled pore glass (CPG) or reverse-phase silica.Support Structure can be globule, sphere, particle, particulate, gel or the form on surface.Surface can be plane, substantially planar Or it is nonplanar.Support can be it is porous or non-porous, and can have expansion or unexpansive characteristic.It can be by support It is shaped to comprising one or more holes, recess or other reservoirs, container, feature or position.Multiple supports can be configured Different location in array.Support is optionally addressable (for example, the robot for reagent delivers), or passes through detection Means include being collected by the scanning of laser irradiation and copolymerization coke or deflect light.Amplification support (such as globule) can be placed in separately On (such as in the hole of the second support) within one support or.

In embodiments, solid support is " particle ", " globule ", " microballon " etc. (optionally but not necessarily in shape It is spherical on shape), with 50 microns or smaller, preferably 10 microns or smaller, 3 microns or smaller, about 1 micron or smaller, about 0.5 micron or smaller, for example, about 0.1,0.2,0.3 or 0.4 micron, or smaller (for example, it is lower than 1 nanometer, about 1-10 nanometers, about 10-100 nanometers, or about 100-500 nanometers) minimum cross-section length (such as diameter).Particle (such as from Dynal, The Dynabead of Oslo, Norway) it can be made of a variety of inorganic or organic material, the material includes but is not limited to glass (example Such as controlled pore glass), silica, zirconium, the polystyrene of crosslinking, polyacrylate, polymethyl methacrylate, titanium dioxide, Latex, polystyrene etc..Magnetization can help the reagent (such as polynucleotides or ligase) of particle connection after amplification Collection and concentration, and can also help other step (such as washing, reagent removal etc.).In certain embodiments of the present invention In, the Particle Swarm with different shape size and/or color can be used.Optionally for example with quantum dot encoding microsomal so as to It can individually or uniquely identify such particle.

In some embodiments, bead surface can be functionalized to connect multiple the first primers.In some embodiments In, globule can be any size that can be assembled in reaction chamber.For example, a globule can be assembled in reaction chamber.Some In embodiment, more than one globule can be assembled in reaction chamber.In some embodiments, the minimum cross-section length of globule (such as diameter) can be about 50 microns or smaller, or about 10 microns or smaller, or about 3 microns or smaller, about 1 micron or more It is small, about 0.5 micron or smaller, for example, about 0.1,0.2,0.3 or 0.4 micron, or it is smaller (for example, about 1-10 receives lower than 1 nanometer Rice, about 10-100 nanometers, or about 100-500 nanometers).

In some embodiments, globule can be connected with multiple one or more different primer sequences.In some implementations In scheme, globule can be connected with a kind of multiple primer sequences, or can be connected with two or more multiple different primer sequences. In some embodiments, globule can be connected at least 1,000 primer, or about 1,000-10,000 primer, or about 10, 000-50,000 primer, or about 50,000-75,000 primer, or about 75,000-100,000 primer or more it is multiple Primer.

In some embodiments, reaction mixture includes recombinase.Recombinase may include can induce recombination event or Increase any reagent of the occurrence frequency of recombination event.Recombination event includes that the different polynucleotide chain of two of them recombinates each other Any event.Recombination may include homologous recombination.Recombinase can be enzyme or its genetically engineered derivative.Recombinase can appoint Selection of land associates (such as combination) with single-stranded oligonucleotide (such as the first primer).In some embodiments, it is catalyzed homologous recombination Enzyme can by combine single-stranded oligonucleotide formed nucleoprotein complex.In some embodiments, homologous recombination enzyme is as core The analogous parts of the combinable double-stranded polynucleotide in albumen composition hair part.In some embodiments, polynucleotides is homologous Part can be hybridized to the first primer at least partly.In a certain embodiment, the analogous parts of polynucleotides can partly or Fully it is complementary to the first primer at least partly.

In some embodiments, homologous recombination enzyme can be by forming nucleoprotein complex and being bound to double-stranded polynucleotide Analogous parts invade the (U.S. of Zarling to form recombination intermediate (D- ring is formed) Lai Cuihua chain with triple strand structure The patent No. 5,223,414, the U.S. Patent number 5,273,881 and 5,670,316 of Sena, and U.S. Patent number 7,270,981, 7,399,590,7,435,561,7,666,598,7,763,427,8,017,339,8,030,000,8,062,850 and 8071308).In some embodiments, homologous recombination enzyme includes wild type, mutant, recombinant, fusion or its segment. In some embodiments, homologous recombination enzyme include from it is any biology include Myoviridae (myoviridae) (such as from UvsX, RB69 etc. of Bacteriophage T4), the enzyme of Escherichia coli (such as recA) or people (such as RAD51).

It in some embodiments, include one or more auxilins for the method for nucleic acid amplification.For example, auxiliary egg The white activity (authorizing United States Patent (USP) 8,071,308 of Piepenburg etc.) for improving recombinase.In some embodiments, auxiliary Albumen is helped in combination with the single-stranded of nucleic acid or recombinase can be loaded on nucleic acid.In some embodiments, auxilin includes Wild type, mutant, recombinant, fusion or its segment.In some embodiments, auxilin can from be used into Any combination of the identical or different species of the recombinase of row nucleic acid amplification reaction.Auxilin can derive from any bacteriophage Body includes myovirus bacteriophage.The example of myovirus bacteriophage includes T4, T2, T6, Rb69, Aeh1, KVP40, non-lever Pseudomonas bacteriophage 133, Aeromonas bacteriophage 65, cyanophage P-SSM2, cyanophage PSSM4, blue-green alge Bacteriophage S-PM2, Rb14, it Rb32, Aeromonas bacteriophage 25, vibrio phage nt-1, phi-1, Rb16, Rb43, bites Thallus 31, bacteriophage 44RR2.8t, Rb49, bacteriophage Rb3 and bacteriophage LZ2.Auxilin can derive from any bacterial species Including Escherichia coli, solfataricus genus (such as sulphur mine sulfolobus solfataricus) or Methanococcus (such as Methanococcus jannaschii).

It in some embodiments, may include single strand binding protein for the method for nucleic acid amplification.Single strand binding protein packet Include myovirus gp32 (such as T4 or RB69), Sso SSB for carrying out bin cure mine sulfolobus solfataricus, the MjA from Methanococcus jannaschii SSB or Escherichia coli SSB albumen.

It in some embodiments, may include that the recombinase that can increase on nucleic acid is loaded into for the method for nucleic acid amplification Albumen.For example, it includes UsvY albumen (United States Patent (USP) 8,071,308 for authorizing Piepenburg) that recombinase, which is loaded into albumen,.

It in some embodiments, may include the albumen of at least one combination nucleic acid for the method for nucleic acid amplification, including Unlock the albumen (such as unwindase) of double-strandednucleic acid.

It in some embodiments, may include at least one for recombinase or polymerization enzyme activity for the method for nucleic acid amplification The confactor of property.In some embodiments, confactor includes one or more bivalent cations.The reality of bivalent cation Example includes magnesium, manganese and calcium.

In some embodiments, can inhibit premature reaction originate under conditions of precincubation nucleic acid amplification reaction.Example Such as, one or more components of nucleic acid amplification reaction can be detained from reaction vessel to prevent premature reaction from originating.In order to start Reaction, can be added bivalent cation (such as magnesium or manganese).It in another example, can be in the at a temperature of precincubation of inhibitory enzyme activity Nucleic acid amplification reaction.It precincubation can react at about 0-15 DEG C or about 15-25 DEG C to inhibit premature reaction to originate.It then can be Incubation reaction is at higher temperature to cause enzymatic activity.

It in some embodiments, may include at least one recombinase on nucleic acid for the method for nucleic acid amplification Assembly or the confactor matched for homologous nucleic acid.In some embodiments, confactor includes any type of ATP packet Include ATP and ATP γ S.

It in some embodiments, may include at least one confactor for generating ATP for the method for nucleic acid amplification.Example If confactor includes converting ADP to the enzyme system of ATP.In some embodiments, confactor include phosphocreatine and Creatine kinase.

In some embodiments, any nucleic acid amplification method disclosed herein can be in the expansion of isothermal or substantially isothermal It is carried out under the conditions of increasing or may include the step of progress under the described conditions.In some embodiments, isothermal duplication condition includes Undergo the nucleic acid amplification reaction of such temperature change: at least certain a part (or entire amplification of the temperature change in amplification Process) period is confined in limited range, including such as temperature change is equal to or less than about 20 DEG C, or about 10 DEG C, or about 5 DEG C, or about 1-5 DEG C, or about 0.1-1 DEG C, or it is less than about 0.1 DEG C.

In some embodiments, can carry out isothermal nucleic acid amplification reaction about 2,5,10,15,20,30,40,50,60 or 120 minutes.

In some embodiments, can be at about 15-25 DEG C, or about 25-35 DEG C, or about 35-40 DEG C, or about 40-45 DEG C, or Isothermal nucleic acid amplification reaction is carried out at about 45-50 DEG C, or about 50-55 DEG C, or about 55-60 DEG C.

It in some embodiments, include multiple and different polynucleotides for the method for nucleic acid amplification.In some implementations In scheme, multiple and different polynucleotides include the mixture of single-stranded or double-stranded polynucleotides or both.In some embodiments In, multiple and different polynucleotides include the polynucleotides of sequence having the same or different.In some embodiments, more A different polynucleotides include the polynucleotides of length having the same or different.In some embodiments, it is multiple not Same polynucleotides include about 2-10, or about 10-50, or about 50-100, or about 100-500, or about 500-1,000, or about 1, 000-5,000, or about 10³–10⁶, or about 10⁶-10¹⁰A or more different polynucleotides.In some embodiments, more A different polynucleotides include the polymer of deoxynucleotide, ribonucleotide and/or its analog.In some embodiments In, multiple and different polynucleotides include naturally occurring, synthesis, recombination, clone, amplification, not expanding or return Shelves (such as preservation) form.In some embodiments, multiple and different polynucleotides include DNA, cDNA, RNA or embedding Close RNA/DNA and nucleic acid analog.

In some embodiments, multiple and different polynucleotide templates may include having to be connected with nucleic acid linker sequence The double-stranded polynucleotide library construction body of one or two end.For example, polynucleotides library construction body may include first and Two ends, wherein first end is connected to the first nucleic acid linker.Polynucleotides library construction body, which may also include, is connected to the second core The second end of sour connector.First and second connectors can be having the same or different sequence.In some embodiments, first Or second at least partly (part i.e. as polynucleotides library construction body) of nucleic acid linker the first primer can be hybridized to.One In a little embodiments, homologous recombination enzyme as nucleoprotein complex part can with first or second nucleic acid linker sequence Polynucleotides library construction body combines.

In some embodiments, polynucleotides library construction body can include changing suitable for any kind of microarray dataset Learn degradation, chain termination, synthesis order-checking, pyrophosphate, large-scale parallel, ion-sensitive and unimolecule platform.

It in some embodiments, include dilution and globule (for example, being connected with multiple first for the method for nucleic acid amplification The globule of primer) reaction polynucleotides amount to reduce the percentage of the globule reacted with more than one polynucleotides.One In a little embodiments, nucleic acid amplification reaction, the polynucleotides pair can be carried out to globule ratio using such polynucleotides Globule ratio is selected to optimize the percentage of the globule of monoclonal polynucleotides group attached thereto.For example, can be with Any one polynucleotides within the scope of about 1:2 to 1:500 carry out nucleic acid amplification reaction to globule ratio.In some embodiments In, polynucleotides include about 1:2, or about 1:5, or about 1:10, or about 1:25, or about 1:50, or about 1:75 to globule ratio, or About 1:100, or about 1:125, or about 1:150, or about 1:175, or about 1:200, or about 1:225, or about 1:225, or about 1: 250.In some embodiments, nucleic acid amplification reaction can produce the globule for not having the polynucleotides for being connected to it, has and connects It is connected to other globules of its a type of polynucleotides and the polynucleotides of more than one type attached thereto Other globules.

In some embodiments, reaction mixture includes one or more primers.For example, reaction mixture may include to Few first Oligonucleolide primers.In some embodiments, the first primer may include being hybridized to a chain of polynucleotides extremely Least a portion of forward direction amplimer.In some embodiments, the first primer includes extendible 3 ' for nucleotide polymerization End.

It in some embodiments, include the other primer for being hybridized to template for the method for nucleic acid amplification.For example, the Two primers can be at least part of reversed amplimer for being hybridized to a chain of polynucleotides.In some embodiments, Second primer includes extendible 3 ' end.In some embodiments, the second primer is not attached to surface.

In some embodiments, third primer can be at least part of forward direction for being hybridized to a chain of polynucleotides Amplimer.In some embodiments, third primer includes extendible 3 ' end.In some embodiments, third is drawn Object is not attached to surface.In some embodiments, third primer includes binding partners or the parent of the nucleic acid for Enrichment Amplification With part (such as biotin).

In some embodiments, primer (such as first, second, and third primer) includes single-stranded oligonucleotide.

In some embodiments, primer at least partly can be at least one chain of polynucleotides in reaction mixture Partial hybridization.For example, primer can at least partly hybridize with the nucleic acid linker for one or two end for being connected to polynucleotides. In some embodiments, primer at least partly can partially or even wholly be complementary to the part of polynucleotides or nucleic acid connects Head.In some embodiments, primer can include chemical degradation, chain termination, synthesis suitable for any kind of microarray dataset Sequencing, pyrophosphate, large-scale parallel, ion-sensitive and unimolecule platform.

In some embodiments, primer (such as first, second or third primer) can have not and in reaction mixture 5 ' or the 3 ' of the partial hybridization of at least one chain of polynucleotides are prominent tail portion (magnetic tape trailer primer).In some embodiments, band Tail primer can have any length, the length including 1-50 or more nucleotide.

In some embodiments, primer includes the polymer of deoxynucleotide, ribonucleotide and/or its analog. In some embodiments, primer includes shape that is naturally occurring, synthesis, recombination, clone, amplification or not expanding Formula.In some embodiments, primer includes DNA, cDNA, RNA or chimeric rna/dna and nucleic acid analog.

In some embodiments, primer can be any length, including about 5-10 nucleotide, or about 10-25 core Thuja acid, or about 25-40 nucleotide, or about 40-55 nucleotide or longer.

It in some embodiments, may include one or more different polymerases for the method for nucleic acid amplification.One In a little embodiments, polymerase include the polymerization that can be catalyzed nucleotide and/or nucleotide analog any enzyme or its segment or Subunit.In some embodiments, polymerase needs extendible 3 ' end.For example, polymerase needs the end of nucleic acid primer 3 ' OH are polymerize with initiation nucleotide.

Polymerase includes any enzyme that can be catalyzed polymerization of the nucleotide (including its analog) into nucleic acid chains.Normally but Not necessarily such nucleotide polymerization can be occurred with template dependent manner.In some embodiments, polymerase can be with It is high fidelity polymerase.Such polymerase may include but be not limited to naturally occurring polymerase and its any subunit and truncation Body, mutated polymerase, variant polymerase, recombination, fusion or the polymerization of the polymerase, chemical modification that are engineered in other ways Enzyme, the molecule of synthesis or assembly and its any analog, derivative or piece for remaining the such ability polymerizeing of catalysis Section.Optionally, polymerase can be the mutated polymerase comprising one or more mutation, and the mutation includes one or more Insertion or missing or two or more polymerizations of the amino acid by other amino acid replacements, one or more amino acid from polymerase The connection of the part of enzyme.As used herein, belong to " polymerase " and its variant and also refer to the portion for containing at least two and being connected to each other The fusion protein divided, wherein first part includes that can be catalyzed the peptide of polymerization of the nucleotide into nucleic acid chains and be connected to second Point, the second part includes the second polypeptide, such as reporter enzyme or processivity promote enzyme.Normally, polymerase includes The catalysis that nucleotide combines and/or nucleotide combines can occur on it for one or more active sites.In some embodiments In, polymerase includes or lacks other enzymatic activity such as 3 ' to 5 ' exonuclease activities or 5 ' to 3 ' exonuclease enzyme activity Property.In some embodiments, it can be separated from cell or generate polymerase using recombinant DNA technology or chemical synthesis process.? In some embodiments, polymerase can be expressed in protokaryon, eukaryon, virus or phage biocontrol.In some embodiments, gather Synthase can be the albumen or its segment of posttranslational modification.

In some embodiments, polymerase may include the bioactivity piece of any one or more polymerases or polymerase Section, be described in Davidson et al. the U.S. Patent Publication No. 2011/0262903 disclosed on October 27th, 2011 and/ Or in the International PCT publication number WO 2013/023176 disclosed on 2 14th, 2013 of Vander Horn et al..

In some embodiments, polymerase can be archaeal dna polymerase and including but not limited to DNA of bacteria polymerase, true Core biology archaeal dna polymerase, archael DNA polymerase.Viral dna polymerase and phage DNA polymerase.

In some embodiments, polymerase can be replicase, DNA dependence polymerase, primase, RNA dependence Polymerase (such as reverse transcriptase of the archaeal dna polymerase including RNA dependence), thermal instability polymerase or thermal stability polymerization Enzyme.In some embodiments, polymerase can be any family A or Type B polymerase.Family A (such as Escherichia coli Pol I), B (such as Escherichia coli Pol II), C (such as Escherichia coli Pol III), D (such as wealthy archeobacteria Pol II), X (such as People Pol β) and Y (such as Escherichia coli UmuC/DinB and eucaryote RAD30/ xeroderma pitmentosum variant) polymerase many Type specification is in Rothwell and Watsman 2005Advances in Protein Chemistry 71:401-440 In.In some embodiments, polymerase can be T3, T5, T7 or SP6RNA polymerase.

In some embodiments, can using a seed type or mixture for polymerase, recombinase and/or ligase come into Row nucleic acid amplification reaction.In some embodiments, nucleic acid amplification can be carried out using low fidelity or high fidelity polymerase Reaction.

It in some embodiments, can be by thermostabilization or thermal instability polymerase come catalytic nucleic acid amplified reaction.

In some embodiments, polymerase can lack 5 ' -3 ' exonuclease activities.In some embodiments, gather Synthase can have strand-displacement activity.

In some embodiments, extinct plants and animal archaeal dna polymerase can be but not limited to thermal stability or thermophilic DNA polymerization Enzyme is for example: bacillus subtilis (Bacillus subtilis) (Bsu) DNA polymerase i large fragment；Thermus aquaticus (Taq) Archaeal dna polymerase；Filamentous Thermus (Thermus filiformis) (Tfi) archaeal dna polymerase；Phi29DNA polymerase；Thermophilic rouge Fat bacillus (Bst) archaeal dna polymerase；9 ° of N-7DNA polymerases of hyperthermophilic archaeal (Thermococcus sp.)；Shi Shi gemma Bacillus (Bacillus smithii) (Bsm) archaeal dna polymerase large fragment；Beach is thermophilic coccus (Thermococcus Litoralis) (Tli) archaeal dna polymerase or Vent^TM(exo-) archaeal dna polymerase (coming from New England Biolabs)；Or " Deep Vent " (exo-) archaeal dna polymerase (New England Biolabs).

It in some embodiments, may include the nucleotide of one or more types for the method for nucleic acid amplification.Nucleosides Acid combines polymerase including alternative or can be by any compound of polymerization enzymatic polymerization.It is normally but nonessential, nucleotide It is later polymerizeing into nucleic acid chains by the nucleotide of polymerase with the selective binding of polymerase；However occasionally nucleotide Can be from polymerization enzymatic hydrolysis without being incorporated in nucleic acid chains, which is herein referred to as " unproductive " event.Such nucleosides Acid not only includes that naturally occurring nucleotide further includes alternative combining polymerase or can be by any similar of polymerization enzymatic polymerization Object (no matter its structure).Although naturally occurring nucleotide generally comprises base, sugar and phosphate moiety, present disclosure Nucleotide may include the compound for lacking any, some or all of such part.In some embodiments, nucleotide Optionally including phosphorus atoms chain, it includes 3,4,5,6,7,8,9,10 or more phosphorus atoms.In some embodiments, phosphorus Chain can be connected to any carbon such as 5 ' carbon of saccharide ring.Using the O or S of insertion by phosphorus chain link to sugar.In an embodiment In, one or more phosphorus atoms in chain can be the part of the phosphate group with P and O.In another embodiment, may be used Utilize O, NH of insertion, S, methylene, substituted methylene, ethylene, substituted ethylene, CNH₂、C(O)、C(CH₂)、CH₂CH₂Or C(OH)CH₂R (wherein R can be 4- pyridine or 1- imidazoles) links together the phosphorus atoms in chain.In an embodiment In, the phosphorus atoms in chain can have comprising O, BH₃Or the side group of S.In phosphorus chain, have the phosphorus atoms of side group in addition to O can be with It is the phosphate group replaced.In phosphorus chain, there are the phosphorus atoms of the atom of the insertion in addition to O can be substituted phosphate group. Some examples of nucleotide analog are described in the U.S. Patent number 7,405,281 of Xu.

The some examples that can be used for the nucleotide of disclosed method and composition include but is not limited to ribonucleotide, deoxidation Ribonucleic acid, the ribonucleotide of modification, the DNA of modification, ribonucleotide polyphosphate, DNA Polyphosphate, the ribonucleotide polyphosphate of modification, the DNA polyphosphate of modification, peptide nucleotide, modification Peptide nucleotide, metal nucleosides, phosphonate ester nucleosides and the phosphate of modification-sugar backbone nucleotide, aforesaid compound analog, Derivative or variant etc..In some embodiments, nucleotide may include the α phosphate and sugared or core for replacing bridge joint nucleotide Between α the and β phosphate of thuja acid or any other two phosphate of the β and γ phosphate of nucleotide or nucleotide or its The non-oxygen part such as sulphur-of any combination of oxygen part or borine-part.

In some embodiments, nucleotide is unlabelled.In some embodiments, nucleotide include label and Herein referred to as " nucleotide of label ".In some embodiments, label can be any part for being connected to nucleotide The fluorescence of (being the phosphate group farthest from sugar including base, sugar or any phosphate group intervened therebetween or terminal phosphate group) The form of dyestuff.

In some embodiments, nucleotide (or its analog) can be connected to label.In some embodiments, it marks Including optically detectable part.In some embodiments, label includes being generally not present in naturally occurring nucleotide In part.For example, label may include fluorescence, shine or radioactive part.

In some embodiments, it may also include enriching step for the method for nucleic acid amplification.In some embodiments, Method for nucleic acid amplification can produce at least one and be connected with multiple multicores with the sequence complementary with template polynucleotide The globule of thuja acid (such as nucleic acid of amplification).At least one for being connected to the polynucleotides of globule can be hybridized to biotinylation Partial polynucleotides (such as reversed amplified production with third primer).In some embodiments, enriching step includes By by the polynucleotides with biotin moiety and purifying globule (such as the paramagnetic for being connected to streptavidin part Globule) (such as the MyOne from Dynabeads^TMBead) in conjunction with forming purifying compound.In some embodiments, may be used Compound will be purified from reaction mixture separation/removal by the attraction using magnet.

In some embodiments, the amplicon of the nucleic acid group comprising substantially monoclonal is respectively placed, distributed or determined Different loci in site array.

In some embodiments, disclosed method includes by single template molecule (such as the single target multicore glycosides of sample Acid) distribution, place or in other ways positioning in reaction chamber or site (such as in array).It can be by making with multicore glycosides The fluid of acid sample flows through reaction chamber and distributes single polynucleotides into reaction chamber from sample.It distributes single into reaction chamber Polynucleotides can be single-stranded or double-strand.In some embodiments, it is expanded in reaction chamber or site after the distribution Nucleic acid.

In some embodiments, different single target polynucleotides can not distributed to arrangement in an array not from sample In each of same reaction chamber.It can be by making the fluid with polynucleotides sample flow through reaction chamber for different single multicores Thuja acid is distributed from sample into each of different reaction chambers.Distribute the different lists into each of different reaction chambers A polynucleotides can be single-stranded or double-strand.

In some embodiments, method includes distributing single polynucleotides into reaction chamber, and expand in reaction chamber Increase single polynucleotides, to generate monoclonal nucleic acid group in the reaction chamber.

In some embodiments, for single target polynucleotide being distributed into reaction chamber and being expanded single target multicore glycosides The method of acid includes sample of nucleic acid.In some embodiments, it can be distributed from the sample of nucleic acid with multiple polynucleotides single Polynucleotides or different single polynucleotides.For example, sample of nucleic acid may include about 2-10, or about 10-50, or about 50-100, Or about 100-500, or about 500-1,000, or about 1,000-5,000, or about 10³-10⁶, or more different polynucleotides. Different polynucleotides can be having the same or different sequence.Different polynucleotides can be having the same or different length Degree.Sample may include the mixture of single-stranded or double-strand polynucleotides or both.

In some embodiments, for single target polynucleotide being distributed into reaction chamber and being expanded single target multicore glycosides The method of acid includes distributing single polynucleotides.In some embodiments, single polynucleotides may include single-stranded and double-strand Nucleic acid molecules.In some embodiments, nucleic acid may include the poly- of deoxynucleotide, ribonucleotide and/or its analog Close object.In some embodiments, nucleic acid may include it is naturally occurring, synthesis, recombination, clone, amplification, do not expand Or filing (such as preservation) form.In some embodiments, nucleic acid may include DNA, cDNA, RNA or chimeric RNA/ DNA and nucleic acid analog.In some embodiments, single polynucleotides may include being included in one or two end to be connected to The nucleic acid library construct of the nucleic acid of oligonucleotide joint.In some embodiments, nucleic acid library construct can be suitable for Any kind of microarray dataset include chemical degradation, chain termination, synthesis order-checking, pyrophosphate, large-scale parallel, ion-sensitive and Unimolecule platform.

In some embodiments, the site of array may include that one or more reaction chambers (can be on the surface of solids Hole).Reaction chamber can have the hole for limiting width and depth.The scale of reaction chamber can be it is enough with allow reagent deposition or For being reacted.It includes cylindrical, polygon or combination of different shapes that reaction chamber, which can have any shape,.Reaction chamber is appointed He Bike has smooth or irregular surface.Reaction chamber may include the bottom on concave or convex surface that is having plane.Reaction The bottom of room and side wall may include same or different material and/or can be coated with chemical group that the chemical group can be with life Object molecule such as nucleic acid, albumen or enzyme reaction.

In some embodiments, reaction chamber can be aligned in one of multiple reaction chambers in grid or array.Battle array Column may include two or more reaction chambers.Multiple reaction chambers randomly can be arranged or be arranged in orderly array.Orderly Array may include with arrange or with row and column two-dimensional grid arrangement reaction chamber.

Array may include any amount of reaction chamber for depositing reagent and carrying out multiple individual reactions.For example, array It may include at least 256 reaction chambers, or at least 256,000, or at least 1-3 million, or at least 3-5 million, or at least 5-7 hundred Ten thousand, or at least 7-9 million, at least 9-11 million, at least million reaction chambers of 11-13 or even higher density include 13- 700000000 reaction chambers or more.The reaction chamber being arranged in grid can have less than about 10 microns or be less than about 5 microns or Centre distance (such as pitch (pitch)) between adjacent reaction room less than about 1 micron or less than about 0.5 micron.

Array may include the reaction chamber with any width and depth dimension.For example, reaction chamber can have adaptation single micro- The scale of grain (such as microballon) or multiple particles.Reaction chamber can accommodate the water volume of 0.001-100 picoliters.

In some embodiments, at least one reaction chamber can be coupled to one or more sensors or can be welded on one On a or multiple sensors.The reaction chamber for being coupled to sensor can provide the closing for being deposited on reagent therein so as to next reflexive The product answered can be detected by sensor.Sensor can detect the variation of the product from any kind of reaction, the reaction packet Include any nucleic acid reaction such as primer extend or or nucleotide be incorporated to reaction.Sensor can detect ion (such as hydrogen ion), The variation of proton, phosphate group such as pyrophosphate group.In some embodiments, at least one reaction chamber can be coupled to one A or multiple ion-sensitive field effect transistors (ISFET).The example for being coupled to the array of the reaction chamber of ISFET sensor can See U.S. Patent number 7,948,015 and U.S. serial 12/002,781.

In some embodiments, it is (and relevant that amplification method described herein can be carried out in the array of reaction chamber Composition, system and device), wherein the reaction chamber of array forms the part of single fluid system.In some embodiments, The array of multiple reaction chambers may include fluidity interface, and the fluidity interface makes fluid (such as liquid or gas) with controlled Laminar flow flows through reaction chamber.In some embodiments, the array of reaction chamber can include the fluid for laminar flow on reaction chamber Pressure head space.In some embodiments, the array of reaction chamber can be the part of flow cell or flow chamber, wherein reaction chamber that It is in fluid communication around here.For example, fluid can be flowed on array so that partially or fully filling the one or more of array Reaction chamber.In some embodiments, fluid can be completely filled with multiple reaction chambers, and excessive fluid can flood the top of reaction chamber Portion is to form fluid layer in the top of reaction chamber.Fluid layer above reaction chamber can provide fluid for multiple reaction chambers in array Connection.In some embodiments, the fluid communication in array between multiple reaction chambers can be used for carrying out in multiple reaction chambers Isolated parallel reaction.For example, be in fluid communication can be used for for polynucleotides and/or reagent being delivered to multiple reaction chambers it is flat to carry out Capable nucleic acid amplification reaction.

In some embodiments, it can will be applied to flow chamber with the sample of multiple and different polynucleotides with will be single Polynucleotides distribute each reaction chamber into array.In some embodiments, other reagent can be applied to flow chamber With distribution in each reaction chamber into array.For example, other reaction reagent may include that particle, one or more enzymes, enzyme are auxiliary Help the factor, primer and/or nucleoside triphosphate.It in some embodiments, can in any order, including continuously or substantially Polynucleotides and reagent, are delivered to the array of reaction chamber by combination simultaneously or both.For example, in some embodiments, The array that can first distribute polynucleotides to reaction chamber then distributes reagent, or opposite sequence can be used, or can be substantially Simultaneously distribute polynucleotides and reagent.

In some embodiments, any method (including flow chamber) can be used to be delivered to polynucleotides and/or reagent The reaction chamber of certain percentage in array.For example, the percentage for the reaction chamber being loaded in array includes about 1-25%, or about 25%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70% or higher percentage.In some embodiment party In case, the reaction for being loaded with polynucleotides and/or reagent can be increased by carrying out the loading step of two or more bouts The percentage of room.For example, polynucleotides and/or reagent can be distributed multiple reaction chambers into array in first leg by (a), (b) in second leg, polynucleotides and/or reagent can be distributed to identical array.It can carry out additional loading bout (example Such as third, 4th or more bout).In some embodiments, it can be carried out between any loading bout any kind of It reacts and/or any kind of reaction can be carried out after the completion of multiple loading bouts.For example, can between any loading bout into Row nucleic acid amplification reaction can carry out nucleic acid amplification reaction after the completion of multiple loading bouts.In some embodiments, every After a loading bout, polynucleotides and globule can be prevented to migrate out reaction chamber on array compound laying.For example, It, can be by the solution laying comprising at least one screening agent on array after each loading bout.In some embodiments, it sieves Dividing agent includes cellulose derivative.Alternatively, can be by oil reservoir laying in the hole of array or the top of indoor reaction mixture.

In some embodiments, disclosed method (and relevant composition, system and kit) further includes expanding By the nucleic acid-templated site for being connected to array before template.Optionally, site includes primer, and the connection includes by primer It is hybridized to the primer binding site of template.For example, the site in array may include at least one primer fixed, the primer packet Containing at least partly complementary sequence for the primer binding site of template for being assigned or being placed at site.Primer promotes template extremely The connection of array.In some embodiments, most of site in array includes at least one primer.The primer of different loci It can be mutually the same.Alternatively, the primer of different loci can be it is different from each other.In an exemplary embodiment, At least two sites respectively include different target specific primers.

Any method appropriate can be used that primer is connected to the site of array.Primer is connected to receiving for reaction chamber by particle The surface of rice array (for example, the ISFET array type for being used for the sequencing based on ion), first at least some reactions of array Indoor synthesis or prepare three dimensional matrix can be it is useful.It in embodiments, can be by polymer substrate precursor applications Yu Yuyi The array in a or multiple united holes of sensor.Polymer substrate precursor can be polymerize to form the array of polymer substrate.This It includes hereditary sequencing technologies that a little polymer substrates, which can be conjugated in oligonucleotides and can be used for a variety of analytical technologies,.

In some embodiments, by hydrophilic polymer matrix distribute with sensor (such as the sensing of sensor array Device) in united hole.In instances, hydrophilic matrix is such as hydrogel matrix.Hydrophilic matrix can be with one-to-one structure pair It should be in the sensor of sensor array.In other examples, the sensor of sensor array may include field effect transistor (FET) Sensor, such as ion-sensitive field effect transistor (ISFET).Particularly, host material is in-situ solidifying and is adapted to The structure of a body opening of sensing device.Void area between hole, which can be, there is no polymer substrate.In instances, The bonding surface for being for example covalently bonded to hole of host material.In instances, hole has the depth in 100 nanometers to 10 micron ranges Degree or thickness.In another example, hole can have the characteristic diameter in 0.1 micron to 2 micron ranges.

It, can be by will include that the aqueous solution of polymer precursor is applied in the hole of hole array and to be formed in illustrative methods Polymer substrate.The capacity for the hydrous material that the immiscible fluid being placed on hole array isolation can be used to be limited by hole array. Can starting soln the capacity through being isolated to promote the polymerization of matrix precursor, so as to cause array of substrates of the distribution in hole.? In example, the aqueous solution comprising matrix precursor is distributed to the hole of sensing device by making aqueous precursor flow through hole.Another In a example, it is included in aqueous solution as dispersed phase in emulsion.Dispersed phase can be settled or be excited in the hole of hole array.It can Carry out the polymerization of starting substrate precursor using the initiation factor being placed in water phase or in immiscible fluid.In another example, may be used Hot starting polymerization.

In another illustrative methods, it can sensed by the way that starting molecule is anchored on the surface in the hole of hole array Array of substrates is formed in the hole of device.Solution comprising matrix precursor can be provided on the hole of hole array.Initiation factor can originate The polymerization of matrix precursor causes to form polymer substrate in the hole of hole array.In other examples, the side of the combination above method Face is to further enhance the formation of array of substrates.

In special embodiment, sequencing system include wherein be placed in sensor array flow cell, including with sensing The telecommunication circuit of device array electronic communication, and include that the container being in fluid communication with flow cell and fluid control.In instances, scheme 13 show the expanded view of flow cell 100 and sectional view and show the part of flow chamber 106.Reagent flow 108 flows through hole array 102 surface, wherein reagent flow 108 flows above the open end in the hole of hole array 102.Hole array 102 and sensor array Column 105 can form the integrated unit for constituting the lower wall (or bottom surface) of flow cell 100 together.Reference electrode 104 can be fluidly coupled To flow chamber 106.In addition, flow cell lid 130 encapsulates flow chamber 106 so that reagent flow 108 to be included in limited region.

Figure 14 shows the expanded view in hole 201 and sensor 214 (as shown at the 110 of Figure 13).It can be based on generation Reaction property and the labelling technique (if any) or reagent, by-product that use come the volume, shape, length of selecting hole Width is than (such as bottom width is to hole depth rate) and other size characteristics.Sensor 214 can be chemical field-effect transistor (chemFET), more particularly ion-sensitive FET (ISFET), with floating gate 218, floating gate 218 has optionally Pass through the sensor board 220 separated inside material layer 216 and hole.(do not show in addition, conductive layer can be placed on sensor board 220 Out).In instances, material layer 216 includes ion-sensitive material layer.Material layer 216 can be ceramic layer, for example, except it is other with The nitride of outer zirconium, hafnium, tantalum, aluminium or titanyl compound or titanium.In instances, material layer 216 can have 5nm to 100nm, such as The thickness of 10nm to 70nm, 15nm to 65nm or 20nm to 50nm.

Although material layer 216 is shown as except the boundary for extending in the FET component of display, material layer 216 can be along hole 201 bottom and optionally along hole 201 wall extend.Sensor 214 can incude (and generating relevant output signal) and be present in biography The amount of charge 224 in the opposite material layer 216 of sensor plate 220.The variation of charge 224 can cause 221 He of source electrode of chemFET Curent change between drain electrode 222.In turn, chemFET can be used directly for providing the output signal based on electric current or utilize another Outer circuit provides the output signal based on voltage indirectly.Reactant, washing solution and other reagents can pass through flooding mechanism 240 are movable into and out hole.

In embodiments, the reaction carried out in hole 201 can be the characteristic to identify or measure purpose analyte Or the analysis reaction of property.Such reaction can either directly or indirectly have an impact the quantity of electric charge near sensor board 220 By-product.If such by-product generate in a small amount decay rapidly or with other component reactions, can simultaneously in hole 201 Multiple copies of same analyte are analyzed, to increase the output signal generated.In embodiments, the multiple of analyte can be copied Shellfish is connected to solid support 212 before or after being placed in hole 201.Solid support 212 can be polymer substrate, such as Hydrophilic polymer matrix, such as hydrogel matrix or similar.For simplicity and ease of explanation, solid support 212 is herein In also referred to as polymer substrate.

Hole 201 can be defined by wall construction, and the wall construction can be formed by one or more layers material.In instances, wall construction Can have from the lower surface in hole and extend to 0.01 micron to 10 microns of upper surface, such as 0.05 micron to 10 microns, 0.1 micron To 10 microns, 0.3 micron to 10 microns or 0.5 micron to 6 microns of thickness.Particularly, thickness can be at 0.01 micron to 1 In the range of micron, such as in the range of 0.05 micron to 0.5 micron or 0.05 micron to 0.3 micron.Hole 201 can have No more than 5 microns, such as no more than 3.5 microns, it is no more than 2.0 microns, is no more than 1.6 microns, is no more than 1.0 microns, does not surpass Cross 0.8 micron or the characteristic diameter no more than 0.6 micron, the diameter by 4 multiplied by cross-sectional area (A) divided by the square root of Pi (for example, sqrt (4*A/ π)) Lai Dingyi.In instances, hole 201 can have at least 0.01 micron of characteristic diameter.

Although Figure 14 shows single-wall structure and monolayer material layer 216, system may include one or more wall constructions Layer, one or more conducting shells or one or more material layers.For example, wall construction can be formed by one or more layers, the layer Oxide comprising TEOS or silicon or the nitride comprising silicon.

In the specific example shown in Figure 15, system 300 includes the hole wall structure 302 for defining the array in hole 304, described Hole is placed on the sensing pad of sensor array or is operationally coupled to it.Hole wall structure 302 defines upper surface 306.With hole United lower surface 308 is placed on the sensing pad of sensor array.Hole wall structure 302 defines upper surface 306 and lower surface 308 Between side wall 310.As described above, the material layer contacted with the sensing pad of sensor array can be along the hole of the array in hole 304 At least partly extension for the wall 310 that lower surface 308 or edge are defined by hole wall structure 302.Upper surface 306 can not have material layer.It is special Not, polymer substrate can be placed in the hole of the array in hole 304.Upper surface 306, which can be, there is no polymer substrate. For example, upper surface 306 may include the area of not polymer substrate, such as at least the 70% of the gross area, the gross area is at least 80%, about the 100% of at least 90% or the gross area of the gross area.

Although the wall surface of Figure 14 is shown as substantially perpendicularly and outwardly extends, wall surface can be different Side upwardly extends and can have different shapes.Term " substantially perpendicularly " means orthogonal with the surface defined by sensing pad Component direction on extend.For example, as shown in Figure 16, hole wall 402 can be extended vertically, with the table defined by sensing pad The normal component 412 in face is parallel.In another example, wall surface 404 substantially hangs down on the outward direction for leave sensing pad Directly extend, to provide the hole opening bigger than the following table area in hole.As shown in Figure 16, wall surface 404 with surface The 414 parallel vertical component side of normal component 412 upwardly extends.In alternative example, wall surface 406 is in inward direction On substantially perpendicularly extend, to provide more smaller than the following table area in hole opening area.Wall surface 406 with surface 414 The parallel component direction of normal component 412 on extend.

Although some semiconductors or CMOS manufacturing process, which can lead to, to be had by straight line display surface 402,404 or 406 The structure of non-linear shape.Particularly, wall surface such as wall surface 408 and upper surface such as upper surface 410 in shape can be with It is arc or there can be a various non-linear forms.Although the structure and equipment in company with display are described as having linear layer, table Face or shape, but can be different in some extent by actual layer, surface or the shape that semiconductor technology generates, it may include aobvious The variation of the non-linear and arc for the embodiment shown.

Figure 17 shows that illustrative includes the hole of ion-sensitive material layer.For example, pore structure 502 can define the battle array in hole Column, such as exemplary bore 504,506 or 508.Hole (504,506 or 508) can operationally be coupled to the sensor of lower section (not Display) or it is connected to the sensor of such lower section.Exemplary bore 504 includes to define the bottom in hole 504 and extend to structure 502 In ion-sensitive material layer 510.Although not shown in Figure 17, conducting shell, such as grid, such as ion-sensitive field effect The floating gate of transistor is answered, the lower section of ion-sensitive material layer 510 can be located at.

In another example, as through hole 506 is shown, ion-sensitive material layer 512 can define the bottom in hole 506 And it is not extend in structure 502.In other examples, hole 508 may include the side wall 516 along the hole 508 defined by structure 502 The ion-sensitive layer 514 at least partly extended.Same Shangdi, ion-sensitive material layer 512 or 514 can underlying electronics dresses On the conducting shell or grid set.

In Figure 14, host material 212 and pore structure are conformal.Particularly, host material can in-situ solidifying with hole Wall and bottom surface it is conformal.The upper surface for defining hole may include the area that there is no host material, such as the gross area is at least 70%, at least the 80% of the gross area, about the 100% of at least 90% or the gross area of the gross area.Depending on the property of pore structure, gather Polymer matrix can physically be fixed to hole wall structure.In another example, polymer substrate can be chemically bonded to hole wall knot Structure.Particularly, polymer substrate can covalently be bound to hole wall structure.In another example, hydrogen bond or ionic bond can be passed through Polymer substrate is bound to hole wall structure.

Can be from matrix precursor, such as the monomer that can polymerize completely, such as the monomer based on vinyl, form polymer matrix Matter.Specifically, monomer may include hydrophilic monomer, such as acrylamide, vinyl acetate, hydroxy alkyl methacrylate (hydroxyalkylmethacrylate), its variant or derivative, its copolymer or any combination thereof.In specific example In, hydrophilic monomer is acrylamide, such as functionalized with the acryloyl comprising hydroxyl, amino, carboxyl, halogen or combinations thereof Amine.In instances, hydrophilic monomer is aminoalkyl acrylamides, (D is shown the functionalized acrylamide of POLYPROPYLENE GLYCOL blocked with amine Be shown in hereinafter), propylene piperazine (C, display below) or combinations thereof.In another example, acrylamide can be hydroxyl Alkyl acrylamide, such as hydroxyethyl acrylamide.Specifically, alkylol acrylamides may include N- tri- (methylol) methyl) Acrylamide (A, display is below), N- (methylol) acrylamide (B, display is below) or combinations thereof.At another In example, comonomer may include the acrylate or acrylamide of halogen modification, such as N- (5- acetyl bromide amido amyl) third Acrylamide (BRAPA, E, display is below).In another example, comonomer may include oligonucleotides-modified propylene Acid esters or acrylamide monomer.In other examples, the mixture of monomer, such as alkylol acrylamides and amine function can be used Change the mixture of acrylamide or the mixture of acrylamide and amine-functionalized acrylamide.It in instances, can be in this way Alkylol acrylamides: amine-functionalized acrylamide or acrylamide: the ratio of amine-functionalized acrylamide include amine Functionalized propylene amide, the ratio in the range of 100:1 to 1:1, such as 100:1 to 2:1,50:1 to 3:1,50:1 extremely In the range of 5:1 or 50:1 to 10:1.In another example, can be with such alkylol acrylamides: bromine be functionalized Acrylamide or acrylamide: the ratio of the functionalized acrylamide of bromine includes amine-functionalized acrylamide, and the ratio exists In the range of 100:1 to 1:1, such as in the range of 100:1 to 2:1,50:1 to 3:1,50:1 to 5:1 or 50:1 to 10:1.

A

B

C

D

E

In other examples, it may include the functionalized acrylamide of oligonucleotides or acrylate monomer, such as Acrydite^TMOligonucleotides is incorporated in polymer substrate by monomer.

Another exemplary substrates precursor includes crosslinking agent.In instances, with monomer to the 15:1 to 1:2 of crosslinking agent, example If the mass ratio of 10:1 to 1:1,6:1 to 1:1 or 4:1 to 1:1 includes crosslinking agent.Particularly, crosslinking agent can be divinyl friendship Join agent.For example, divinyl crosslinking agent may include bisacrylamide, such as N, two (2- ethoxys) third of N '-(ethane -1,2- diyl) Acrylamide, N, N '-(2- hydroxypropyl alkane -1,3- diyl) bisacrylamide or combinations thereof.In another example, divinyl crosslinking agent Including ethyleneglycol dimethacrylate, divinylbenzene, hexamethylene bisacrylamide, trimethylol-propane trimethacrylate, Its shielded derivative or combinations thereof.

In one aspect, polymer network includes in the range of having 3-20%, in the range of more preferably 5-10% The polyacrylamide gel of total monomer percentage.In one embodiment, model of the crosslinking agent percentage of monomer in 5-10% In enclosing.In specific embodiments, polymer network includes 10% total acrylamide, wherein 10% is that bisacrylamide is handed over Join agent.

It can be by the initiation factor in solution come starting polymerization.For example, initiation factor can be based on water.At another In example, initiation factor can be hydrophobic initiation factor, be preferably placed in hydrophobic phase.Exemplary initiation factor includes ammonium persulfate Or TEMED (tetramethylethylenediamine).TEMED can accelerate the synthesis speed of the free radical from persulfate, and then catalytic polymerization. Persulfate free radical for example converts free radical for acrylamide monomer, with non-activated monomer reaction with starting polymerization chain Formula reaction.The polymer chain of extension can be randomly crosslinked, and so as to cause with the porous gel of characteristic, the porosity is taken Certainly in polymerizing condition and monomer concentration.Riboflavin (or riboflavin -5 '-phosphate) also is used as the source of free radical, usually It is applied in combination with TEMED and ammonium persulfate.In the presence of light and oxygen, riboflavin is converted into it with starting polymerization Active colorless form, this is commonly referred to as photochemical polymerization.

In another example, nitrogenous initiation factor can be used for starting polymerization.Illustrative water-soluble nitrogenous starting because Son is shown in table 1, and the illustrative nitrogenous initiation factor of oil-soluble is shown in table 2.Particularly, nitrogenous initiation factor can be Azodiisobutyronitrile (AIBN).

Table I

Water-soluble nitrogenous initiation factor compound

Table II

The nitrogenous initiation factor compound of oil-soluble

In other examples, the precursor of polymer substrate may include surfactant additive to enhance and the combination on surface. Exemplary additives include functionalized propylene acid monomers or functionalized acrylamide monomer.For example, acrylic monomers can be through official Energyization is with mating surface material, such as forms the bottom in hole or the ceramic material of side wall.In instances, additive may include propylene Base-phosphonate ester, such as metering system phosphonate ester.In another example, additive may include dimethylacrylamide or poly- two Methacrylamide.In other examples, additive may include being modified by polymerizable group (such as acrylic acid groups) Polylysine.

In another example, atom transfer radical polymerization (ATRP) can be used to promote to polymerize.ATRP system can wrap Include initiation factor, transition metal ions and ligand.Exemplary transition metal ion complex includes the complex compound based on copper.Example Property ligand include 2,2 '-two pyridines,-two pyridine of 4,4 '-two -5- nonyl -2,2 ', 4,4 ', 4 "-three (5- nonyls) -2,2 ': 6 ', 2 "-terpyridyls, N, N, N ', N ', N "-pentamethyl-diethylenetriamine, three second tetramine of 1,1,4,7,10,10- hexamethyl, three (2- dimethylaminoethyl) amine, N, N- bis- (2- picolyl) octadecylamine, N, N, N ', N '-four [(2- pyridine) methyl] ethylenediamine, Three [2- pyridine) methyl] amine, three (2- aminoethyl) amine, three (2- bis- (3- butoxy -3- oxygen propyl group) aminoethyl) amine, three (2- bis- (3- (2- ethyl hexyl oxygen) -3- oxygen propyl group) aminoethyl) amine, three (2- bis- (ten dioxy -3- oxygen propyl group of 3-) aminoethyl) amine, its variant With derivative or combinations thereof.Exemplary initiation factor include 2- bromopropionitrile, ethyl 2- isobutyl bromide ester, ethyl 2 bromopropionic acid ester, Methyl 2 bromopropionic acid ester, 1- phenyl bromoethane, paratoluensulfonyl chloride, 1- cyanogen -1- Methylethyl diethyldithiocar bamic acid Ester, 2- (N, N- diethyl-dithio carbamyl)-ethyl isobutyrate, dimethyl 2,6- dibromo pimelate, its variant spread out Biology and any combination thereof.Optionally, BRAPA monomer can be used as branching agent when there are ATRP system.

In instances, in surface starting ATRP polymer bonds are directly bonded to surface.For example, can transition metal from In the presence of son/ligand complex, by acrylic monomers, acrylamide monomer, Acrydite^TMMonomer, succinimide Acrylate, double acrylic acid or bisacrylamide monomer, its derivative or combinations thereof are applied to the surface of starting in the solution.

In another, ATRP system can be used for using modified phosphonate, sulfonate, silicate or zirconic acid salinization Close the surface that polymer is connected to hole by object.It specifically, can be derivative by amine or hydroxy-end capped phosphonate ester or its alkoxy Object is applied to surface and is originated using initiation factor.Can application of catalyst complex compound and monomer, with extensional surface compound.

In illustrative methods, the aqueous solution of the precursor comprising polymer substrate can be applied to the knot for defining the array in hole In the hole of structure.Can by provided on hole the polymerization of the polymer precursor in the solution in immiscible fluid and starter hole come every From the aqueous solution in hole.

For example, Figure 18 shows the exemplary bore structure 602 for defining hole 604.One or more sensors (not shown) can To be operationally coupled or be connected to hole 604.For example, one or more sensors may include and at least bottom surface in hole 604 electricity The gate structure of communication.It is provided on hole comprising the aqueous solution 606 of polymer precursor in addition to other components and will include poly- The solution of polymer precursor is distributed into hole 604.Exemplary polymer precursor include among other things monomer, crosslinking agent, starting because Son or surfactant, such as described above.Optionally, can use hydrophilic solution before the deposition, for example, comprising water, The solution of or mixtures thereof alcohol, or the solution comprising water and surfactant, to soak hole 604.Exemplary alcohols include isopropanol. In another example, alcohol includes ethyl alcohol.Although not showing, the side wall in the bottom surface in hole and optionally hole may include that ion is quick Ability.Such ion-sensitive material can overlay on lower section electronic device such as field effect transistor conducting structure it On.One or more surfaces in surfactant additive processing hole can be used before applying the solution comprising polymer precursor.

Distribution of the aqueous solution comprising polymer precursor into hole 604 can by shaking structure (such as pass through rotation or whirlpool Rotation) it further strengthens.In another example, vibration such as sound wave or ultrasonic activation can be used to increase aqueous solution in hole Distribution in 604.In other examples, the degasification of vacuum pump device to hole can be used and apply solution under negative gauge pressure.In instances, exist Aqueous solution is distributed to hole at room temperature.In another example, aqueous solution is distributed at a temperature below the room temperature, particularly when making It is so assigned when with the initiation factor based on water.Alternatively, distributing aqueous solution at elevated temperatures.

As shown in Figure 19, immiscible fluid 708 is applied on hole 604, aqueous solution 606 is pushed away to the top in hole Aqueous solution 606 in portion and clearance hole 604, as shown in Figure 20.Exemplary immiscible fluid includes mineral oil, silicone oil (example Such as poly- (dimethyl siloxane)), heptane, carbonic acid ester oil (such as diethylhexyl carbonic ester (Tegosoft)) or A combination thereof.

Initiation factor can be applied in aqueous solution 606.Alternatively, initiation factor can be provided in immiscible fluid 708.It can lead to It crosses change substrate temperature and carrys out starting polymerization.Alternatively, polymerization can occur at room temperature.It specifically, can be by polymer precursor solution It is kept for 10 minutes to 5 hours at 20 DEG C to 100 DEG C, such as at a temperature of 25 DEG C to 90 DEG C, 25 DEG C to 50 DEG C or 25 DEG C to 45 DEG C, Such as 10 minutes to 2 hours or 10 minutes to 1 hour.

Due to polymerization, the array of polymer substrate 912 is formed in the hole 604 defined by pore structure 602, such as Figure 21 Shown.Optionally, array can be washed with NaOH (such as 1N NaOH) to remove polymer from the void area between hole.

It will include the emulsion in immiscible fluid as the aqueous solution of the polymer precursor of dispersed phase in alternate example For by the droplet deposition of aqueous solution in hole.For example, pore structure 1002 defines hole 1004 as shown in Figure 22.Hole can be with Operationally it is coupled or is electrically connected to one or more sensors (not shown).As described above, the bottom table of the wall in hole 1004 Face and optionally side surface can be defined by ion-sensitive material layer, and the material layer can overlay on the conduction of the electronic device of lower section On component.

Emulsion 1006 may include the aqueous droplet comprising polymer precursor dispersed in continuous immiscible fluid 1010 1008.Droplet 1008 can be deposited in hole 1004.Particularly, aqueous droplet 1008 has bigger than immiscible fluid 1010 close Degree.Exemplary immiscible fluid includes mineral oil, silicone oil (such as poly- (dimethyl siloxane)) heptane, carbonic acid ester oil (such as two Ethylhexyl carbonate (Tegosoft)) or combinations thereof.In other examples, rotation, vortex or ultrasound can be passed through Treatment fluid or structure promote distribution of the aqueous droplet into hole 1004.Optionally, hydrophilic solution can be used before the deposition, Such as the solution comprising or mixtures thereof water, alcohol, or the solution comprising water and surfactant, to soak hole 604.Droplet is to hole In distribution during temperature can carry out at room temperature.Alternatively, can be allocated at elevated temperatures.

As shown in Figure 23, droplet merges in hole 1004 to provide the molten of the isolation comprising polymer precursor 1112 Liquid.Optionally, immiscible fluid such as immiscible fluid 1010 or different immiscible fluids without droplet 1008 can be used 1116 replace emulsion 1006.Exemplary immiscible fluid include mineral oil, silicone oil (such as poly- (dimethyl siloxane)) heptane, Carbonic acid ester oil (such as diethylhexyl carbonic ester (Tegosoft)) or combinations thereof.Alternatively, emulsion 1006 can be poly- It is held in place during conjunction.After this manner, the solution 1112 in hole 1004 is kept apart with the solution in other holes 1004.It can originate Polymerization, leads to the polymer substrate 1214 in hole 1004, as shown in Figure 24.As described above, can hot starting polymerization.Another In a example, oily phase initiation factor can be used to carry out starting polymerization.Alternatively, water phase initiation factor can be used to carry out starting polymerization.Especially Ground can apply the second emulsion on hole 1004.Second emulsion may include the water phase of the dispersion comprising water phase initiation factor.

It can be by changing substrate temperature come starting polymerization.Alternatively, polymerization can occur at room temperature.Specifically, can will gather Polymer precursor solution keeps 10 at 20 DEG C to 100 DEG C, such as at a temperature of 25 DEG C to 90 DEG C, 25 DEG C to 50 DEG C or 25 DEG C to 45 DEG C Minute was to 5 hours, such as 10 minutes to 2 hours or 10 minutes to 1 hour.Optionally, it can be washed with NaOH (such as 1N NaOH) Array is to remove polymer from the void area between hole.

In another example, the initiation factor on the surface for being fixed on hole array can be used to form base in the hole of hole array The array of material.For example, structure 1302 can define hole 1304 as shown in Figure 25.Material layer 1306 can define hole 1304 Lower surface.It can will be anchored compound 1308, such as the anchoring compound of atom transfer radical polymerization (ATRP), it is fixed To the material layer 1306 for the bottom surface for defining hole 1304.It is exposed in hole 1304 alternatively, being limited in the hole or layer of structure 1302 Side-wall material can be anchored compound for example as described above be used for ATRP compound.

In such an example, can on the structure 1302 and in hole 1304 application comprising polymer precursor such as monomer, The solution 1310 of crosslinking agent and optionally surfactant additive.Anchoring compound 1308 can be caused to promote from anchoring compound Polymerization is isolated in hole 1304 and polymer is fixed to hole 1304 by the polymerization of extension.In instances, anchoring compound has Surface active groups and distal end free radical form group.Surface active groups may include phosphonate, silicate sulfonate, zirconic acid Salt, titanate or combinations thereof.It may include amine or hydroxyl that distal end free radical, which forms group, the amine or hydroxyl can undergo for example with halogen The transfer of generation (such as bromo) compound simultaneously subsequently forms for polymer, polymer precursor and is anchored into generated polymer The free radical of hole surface.Normally, ATRP system may be selected to terminate after the addition of the monomer of statistically average length or quantity Polymerization.In this way, the polymerization sum in controllable drilling 1304.In other examples, chain extension or termination can be will affect Other reality apply and be added to aqueous solution 1310.

In another example shown in Figure 26, structure 1402 can define hole 1404.Hole 1404 may include along structure 1402 and hole 1404 side wall 1410 extend material layer 1406.Initiation factor1 408 can be along the bottom in hole 1404 and along side wall 1410 It is fixed to material layer 1406.Can apply on structure 1402 and hole 1404 includes polymer precursor, crosslinking agent and other reagents Solution 1412.

As shown in Figure 27, polymer substrate 1512 because starting from the surface in hole 1304 (such as by material layer 1306 definition surfaces) extend polymerization and formed.

It can be by changing substrate temperature come starting polymerization.Alternatively, polymerization can occur at room temperature.Specifically, can will gather Polymer precursor solution keeps 10 at 20 DEG C to 100 DEG C, such as at a temperature of 25 DEG C to 90 DEG C, 25 DEG C to 50 DEG C or 25 DEG C to 45 DEG C Minute was to 5 hours, such as 10 minutes to 2 hours or 10 minutes to 1 hour.

After formation, can activated polymer matrix to promote the conjugation with target analyte such as polynucleotides.For example, can enhance Functional group on polymer substrate is to allow and the combination of target analyte or analyte receptor (such as Oligonucleolide primers).Having In the example of body, available can be the active part that can undergo nucleophilic or parental materials by hydrophilic polymer functional group conversions Reagent modifies the functional group of hydrophilic polymer matrix.For example, can by with sulfonic acid group or chlorine substituted hydroxy at least partly Carry out the hydroxyl in activated polymer matrix.Exemplary sulfonic acid group can from three fluoro ethanesulfonyls (tresyl), methylsulfonyl, Tolysulfonyl or to fluorobenzene sulphonyl (fosyl) chlorine or any combination thereof.Sulphonic acid ester is for allowing nucleophilic group substituted sulfonic acid ester. Sulphonic acid ester can be also reacted with the chlorine being released to provide the chlorination functional group for the process that can be used for being conjugated matrix.In another example In, it can amido in activated polymer matrix.

For example, target analyte or analyte receptor can be bound to hydrophilic polymer by the nucleophilic displacement of fluorine with sulfonic acid group. In specific example, nucleophilic displacement of fluorine can be undergone poly- to replace with the target analyte receptor that nucleophilic group such as amine or sulfydryl block Sulfonic acid group in polymer matrix.Due to activation, the polymer substrate of conjugation can be formed.

In another example, the polymer substrate of sulfonation can further with the single or multiple nucleopilic reagent of single or multiple functionality (such as maleimide) reaction, the connection that the reagent can form with matrix are preserved for the few core comprising electrophilic group simultaneously The nucleophilic reactivity of thuja acid.In addition, parent can be converted by remaining nucleophilic reactivity by being connected to the reagent comprising more electrophilic groups It is electroactive, it will then be connected to the oligonucleotides comprising nucleophilic group.

In another example, the monomer comprising functional group can be added during polymerization.Monomer include for example comprising carboxylic acid, The acrylamide of ester, halogen or other amine reactive groups.Ester group can be hydrolyzed before reacting with few amine (amine oligo).

Other conjugation techniques include using the amine-containing monomer of packet.Amido is can be tried by amine reactivity difunctionality parents' electricity The nucleophilic group that agent is further modified, the reagent generate mono-functional's electrophilic group after being connected to polymer substrate.This The electrophilic group of sample can be reacted with the oligonucleotides with nucleophilic group (such as amine or sulfydryl), lead to the connection of oligonucleotides (by with the electrophilic precursor reactant vacated).

If preparing polymer substrate from the combination of amino-and hydroxy-acrylamide, polymer substrate includes nucleophilic ammonia Base and combination with Neutral hydroxy.Ammonia can be modified using the double electrophilic moiety such as diisocyanate of difunctionality or two-NHS esters Base, so that generating has reactive hydrophilic polymer matrix to nucleophilic group.Exemplary two-NHS ester includes that two-succinyls are sub- Amine acyl C2-C12 alkyl ester, such as two-succinimide acyl suberates or two-succinimide acyl glutarates.

Other activating chemicals include mixing multiple steps to be that adaptation is specific desired by specific functional group conversions Connection.For example, the hydroxyl that sulphonic acid ester is modified can be transformed into nucleophilic group by several method.In instances, sulphonic acid ester with The reaction of azide anion generates the hydrophilic polymer that azide replaces.Azide can be used directly for by can be " CLICK " chemical action carried out in the case where with or without copper catalysis is conjugated to the biomolecule of acetylene substitution.Optionally Ground can be restored by using the catalysis of hydrogen or be converted azide to amine using the reduction of organic phosphine.It may then use that Plurality of reagents such as diisocyanate, two-NHS esters, cyanuric chloride or combinations thereof converts resulting amine to electrophilic group.In reality In example, urea bond (urea linkage) is generated between polymer and connector using diisocyanate, this leads to remaining isocyanide Acid esters group, the biomolecule that can replace with amino are reacted to generate urea bond between connector and biomolecule.Another In a example, amido bond and remaining NHS ester group between polymer and connector, the NHS ester are produced using two-NHS esters The biomolecule that base can replace with amino is reacted to generate amide between connector and biomolecule and connect.In other examples In, amino-triazines key and two remaining chlorotriazine groups between polymer and connector are produced using cyanuric chloride, it is described The biomolecule that one of chlorotriazine group can replace with amino is reacted to generate amino-three between connector and biomolecule Piperazine key.It can be activated by sulfonic acid acyl and other nucleophilic groups are incorporated in matrix.For example, sulfonic acid acyl group matter and thiobenzoate yin from Sulfydryl is incorporated in matrix by the hydrolysis of the Thiobenzoate of reaction and the generation of son, and the sulfydryl then can be with maleimide Substituted biomolecule reaction is connect with generating with sulphur-succinimide of biomolecule.

Sulfydryl can also be reacted with bromo- acetyl group or bromo- amide groups (bromo-amidyl group).In specific example In, when comprising n- (5- acetyl bromide amido amyl) acrylamide (BRAPA) be comonomer when, can by formed for polymerize Thiobenzamide-oligonucleotide compound of bromo- acetyl group reaction on object is to be incorporated to oligonucleotides, such as shows as follows 's.

It can be by reacting following dithiobenzoic acid-NHS compounds with the oligonucleotides that amine blocks and to activate two thio Benzamide-oligonucleotide compound is thio to be formed to form the thiobenzamide-oligonucleotide compound being shown above Benzamide-oligonucleotide compound.

Alternatively, acrydite oligonucleotides can be used to during polymerization to be incorporated to oligonucleotides.Exemplary acrydite is few Nucleotide may include the oligonucleotides of ion exchange.

It can divide on the electrophilic moiety or substrate of the nucleophilic moiety coupling in biomolecule with biology by using on substrate The nucleophilic connection of electrophilic connection coupling on son serves as a contrast to generate biomolecule in refractory material (refractory) or polymeric material Covalent linkage on bottom.Due to the hydrophilic nmature of most of general object biomolecule, being coupled for these for selection is molten Agent is water or the water comprising a certain water-miscible organic solvent to disperse on substrate biomolecule.Particularly, usually in water system Polynucleotides are coupled to substrate in system, because they have polyanion property.Since water is by the way that electrophilic body to be hydrolyzed into For being conjugated inactive part to compete electrophilic body with nucleophilic group, thus aqueous system can lead to the coupling production of low-yield Object, wherein yield is based on couplet electrophilic moiety.When the high yield of the electrophilic moiety of expected response couplet, high concentration Nucleophilic group drive response and slow down hydrolysis, lead to the invalid use of nucleophilic group.In the case where Polynucleotide, phosphorus The alkaline counter-ion of hydrochlorate can be by lipophile balance ionic compartmentation, to help for biomolecule to be dissolved in polarity, anergy In nonaqueous solvents.These solvents may include amide or urea such as formamide, N,N-dimethylformamide, acetamide, N, N- bis- Methylacetamide, hexamethyl-phosphoramide, pyrrolidones, N-Methyl pyrrolidone, N, N, N ', N '-tetramethylurea, N, N '-two Methyl-N, N '-trimethyleneurea or combinations thereof；Carbonate such as dimethyl carbonate, propene carbonate or combinations thereof；Ester such as tetrahydro Furans；Sulfoxide and sulfone such as dimethyl sulfoxide, dimethyl sulfone or combinations thereof；Hinder alcohol (hindered alcohol) such as tert-butyl Ethyl alcohol；Or combinations thereof.Lipophilic cation may include tetra-allkylammonium or four aryl ammonium cations for example tetramethyl-ammonium, tetraethyl ammonium, Tetrapropyl ammonium, tetrabutylammonium, four pentyl ammonium, tetrahexyl ammonium, four heptyl ammoniums, four octyl ammoniums and its alkyl and aryl mixture, four Aryl phosphine cation such as tetraphenylphosphonium, tetraalkyl arsine or four aryl arsines such as four phenylarsines and trialkylsulfonium cation such as three Methyl sulfonium, or combinations thereof.Polynucleotide can be carried out by multiple standards cation switching technology to organic solvent soluble material Conversion (by exchanging metal cation with lipophilic cation).

In specific embodiments, polymer substrate is exposed to target polynucleotide, the target polynucleotide have with It is conjugated in the section of the oligonucleotides complementation of polymer substrate.Make polynucleotides experience amplification, such as passes through polymerase chain reaction Answer (PCR) or recombinase polymeric enzymatic amplification (RPA).For example, target polynucleotide is provided with low concentration, so that single polynucleotides have It is likely located in the single polymer substrate of the array of polymer substrate.Polymer substrate can be exposed to enzyme, nucleotide, salt or its It is enough to promote the component of the duplication of target polynucleotide.

In specific embodiments, enzyme such as polymerase is present in, is connected to or close proximity to polymer substrate.It is more Kind nucleic acid polymerase can be used for method described herein.In an exemplary embodiment, polymerase may include that can be catalyzed multicore The enzyme of the duplication of thuja acid, its segment or subunit.In another embodiment, polymerase can be naturally occurring polymerase, Recombinate polymerase, mutated polymerase, variant polymerase, fusion or the polymerase being engineered in other ways, chemical modification Polymerase, synthetic molecules or its analog, derivative or segment.

In some embodiments, for distributing to reaction chamber and expanding single target polynucleotide single target polynucleotide Method include nucleic acid amplification reaction.In some embodiments, can carry out any kind of nucleic acid amplification reaction includes polymerization Enzyme chain reaction (PCR) (United States Patent (USP) 4,683,195 and 4,683,202 for authorizing Mullis), ligase chain reaction (LCR) (Barany 1991 Proceedings National Academy of Science USA 88:189-193；Barnes 1994 Proceedings National Academy of Science USA91:2216-2220), helicase dependent expand Increase (HDA) or isothermal Autonomous maintenance serial response (1989 Proceedings National Academy of of Kwoh Science USA 86:1173-1177；WO 1988/10315；With United States Patent (USP) 5,409,818,5,399,491 and 5,194, 370)。

In some embodiments, amplified reaction includes recombinase polymeric enzymatic amplification (RPA).(see, for example, Zarling U.S. Patent number 5,223,414, the U.S. Patent number 5,273,881 and 5,670,316 of Sena, and U.S. Patent number 7, 270,981、7,399,590、7,435,561、7,666,598、7,763,427、8,017,339、8,030,000、8,062,850 With 8071308).

In some embodiments, for distributing to reaction chamber and expanding single target polynucleotide single target polynucleotide Method include isothermal duplication condition.In some embodiments, nucleic acid amplification reaction can be carried out under isothermal conditions.Some In embodiment, isothermal duplication condition includes undergoing the nucleic acid amplification reaction of such temperature change: the temperature change is expanding It is confined in limited range during at least certain a part increased, including such as temperature change is at about 20 DEG C, or about 10 DEG C, or about Within 5 DEG C, or about 1-5 DEG C, or about 0.1-1 DEG C, or it is less than about 0.1 DEG C.It in some embodiments, can be in isothermal or thermal cycle Under the conditions of carry out nucleic acid amplification reaction.

In some embodiments, the nucleic acid of content amplification can be used for any nucleic acid sequencing workflow according to this teaching, Including sequencing (such as the SOLiD from Life Technologies for being connected and being detected by oligonucleotide probe^TM, WO 2006/084131), probe-anchoring connection sequencing (such as Complete Genomics^TMOr Polonator^TM), synthesis order-checking (such as Genetic Analyzer and HiSeq from Illumina^TM), pyrophosphate sequencing is (such as from 454Life The Genome Sequencer FLX of Sciences), ion-sensitive sequencing (such as from Ion Torrent Systems, Inc. Personal Genome Machine (PGM^TM) and Ion Proton^TMSequencer) and single-molecule sequencing is flat Platform (such as from Helicos^TMHeliScope^TM)。

In some embodiments, the nucleic acid of content amplification according to this teaching, institute can be sequenced by any sequencing approach Sequencing approach is stated to include synthesis order-checking, include the base that by-product is sequenced using field effect transistor (such as FET and ISFET) detection In the sequencing of ion, chemical degradation sequencing, the sequencing based on connection, sequencing by hybridization, pyrophosphate detection sequencing, capillary electricity Swimming, gel electrophoresis, the next generation, large-scale parallel microarray dataset, detection hydrogen ion or it is other sequencing by-product microarray dataset and Single-molecule sequencing platform.In some embodiments, it at least one that can be used can be hybridized to any portion of polynucleotide constructs The sequencing primer of (including nucleic acid linker or target polynucleotide) is divided to carry out sequencing reaction.

In some embodiments, the method for detecting one or more by-products that nucleotide is incorporated to can be used that root is sequenced The nucleic acid expanded according to this teaching content.Polymerase by detecting the physical chemistry by-product of extension extends detection Pyrophosphate, hydrogen ion, electric charge transfer, heat etc., such as e.g., as disclosed in 7,948,015 He of the U.S. Patent number of Rothberg et al. In the U.S. Patent Publication No. 2009/0026082 (being incorporated herein by reference in their entirety) of Rothberg et al..Detection is based on poly- Other examples of the method for the extension of synthase are found in such as Pourmand et al., Proc.Natl.Acad.Sci., 103: 6466-6470(2006)；Purushothaman et al., IEEE ISCAS, IV-169-172；Anderson et al., Sensors and Actuators B Chem.,129:79-86(2008)；Sakata et al., Angew.Chem.118:2283-2286 (2006)；Esfandyapour et al., U.S. Patent Publication No. 2008/01666727；With Sakurai et al., In Anal.Chem.64:1996-1997 (1992).

The reaction of the generation and the detection that involve ion is widely carried out.This is monitored using direct ion detection method The process of the reaction of sample can simplify many existing bioassay.For example, being generated as by detection by polymerase catalysed core The hydrogen ion for the natural by-product that thuja acid is incorporated to can be monitored to be synthesized by polymerase-mediated template-dependent nucleic acid.Ion-sensitive Sequencing (also referred to as " based on pH's " or " based on ion " nucleic acid sequencing) is utilized ionic byproducts such as hydrogen ion and (is generated The by-product being incorporated to for nucleotide) direct detection.It, can will be in an exemplary system for the sequencing based on ion The trapping nucleic acids being sequenced make nucleotide flow through hole one at a time in micropore, and under the conditions of nucleotide is incorporated to.Polymerase Nucleotide appropriate is incorporated to the chain of growth, the hydrogen ion being released can be changed solution pH, can by with hole be coupled from Sub- sensor detects.The technology does not need the label of nucleotide or the optical module of valuableness, and allows to be sequenced the remote of operation It is remote to complete faster.The example of such method for nucleic acid sequencing and platform based on ion includes Ion Torrent PGM^TMOr Proton^TMSequenator (Ion Torrent^TM Systems,Life Technologies Corporation)。

It in some embodiments, can using the target polynucleotide that the method for this teaching content, system and kit generate As the substrate reacted for biological or chemical, the reaction by sensor include field effect transistor (FET) detect with/ Or monitoring.FET is chemFET or ISFET in different implementation scenarios." chemFET " or chemical field-effect transistor are to use Make the field-effect transistor types of chemical sensor.It is the analogue of mosfet transistor, wherein passing through chemical process To apply the charge on gate electrode." ISFET " or ion-sensitive field effect transistor are used to measure the ion concentration in solution； When ion concentration (such as H+) changes, will correspondingly be changed by the electric current of transistor.The detailed theory of operation ISFET is seen “Thirty years of ISFETOLOGY:what happened in the past 30years and what may Happen in the next 30years, " P.Bergveld, Sens.Actuators, 88 (2003), in pp.1-20.

In some embodiments, FET can be FET array.As used herein, " array " is element such as sensor Or the planar alignment in hole.Array can be one-dimensional or two-dimensional.One-dimensional array, which can be, has a column (or row) in the first dimension Element simultaneously has the array of multiple column (or row) in the second dimension.In the first and the second dimension the number of column (or row) can with or cannot It is identical.FET or array may include 102,103,104,105,106,107 or more FET.

In some embodiments, one or more microfluidic structures can be welded on FET sensor array to mention For the receiving and/or closing of biological or chemical reaction.For example, can configure microfluidic structures to be placed in battle array in an instrument On the one or more sensors of column one or more holes (or micropore or reaction chamber or reacting hole, it is interchangeable herein Ground uses these terms), so as to analyte in given bore one or more sensors detection and measurement given bore placed on it Presence, level and/or concentration.In some embodiments, the corresponding relationship of FET sensor and reacting hole can be 1:1.

Micropore or reaction chamber are usually the hole or hole with clearly defined shape and volume, can be machined in substrate And conventional microbonding connection technology can be used to weld, such as disclosed in following references: Doering and Nishi, Editors,Handbook of Semiconductor Manufacturing Technology,Second Edition (CRCPress,2007)；Saliterman,Fundamentals of BioMEMS and Medical Microdevices (SPIE Publications,2006)；Elwenspoek et al,Silicon Micromachining(Cambridge University Press,2004)；And it is similar.The reality of the structure (such as spacing, shape and volume) of micropore or reaction chamber Example is disclosed in the U.S. Patent Publication 2009/0127589 of Rothberg et al.；The UK Patent Application of Rothberg et al. In GB24611127.

In some embodiments, can contacted with FET such as chemFET or ISFET, operationally coupling or capacitive character Biological or chemical reaction is carried out in the solution or reaction chamber of coupling.FET (or chemFET or ISFET) and/or reaction chamber can be distinguished It is the array of FET or reaction chamber.

In some embodiments, biological or chemical reaction can be carried out in the two-dimensional array of reaction chamber, wherein each anti- Answer room that can be coupled to FET, and each reaction chamber is no more than 10 μm on capacity³(i.e. 1pL).In some embodiments, often A reaction chamber is no more than 0.34pL, 0.096pL or 0.012pL on capacity.Reaction chamber is optionally in the cross-sectional area at top It is upper to be no more than 2,5,10,15,22,32,42,52,62,72,82,92 or 102 square microns.Preferably, array has at least 10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹Or more reaction chamber.In some embodiments, at least one of reaction chamber Operationally it is coupled at least one of FET.

It can be conventional use of according to the CMOS welding technique of conventional cmos welding technique and improvement and in CMOS welding Other semiconductor welding techniques other than technology, to weld for the FET array according to the various embodiments of present disclosure. In addition, various lithographics can be used as the part of array welding procedure.

Example is disclosed in suitable for the exemplary FET array and micropore and fluid of disclosed method and the method for preparing it Such as U.S. Patent Publication No. 20100301398；U.S. Patent Publication No. 20100300895；U.S. Patent Publication No. 20100300559；U.S. Patent Publication No. 20100197507, U.S. Patent Publication No. 20100137143；U.S. Patent Publication Numbers 20090127589；In U.S. Patent Publication No. 20090026082, above-mentioned reference is incorporated herein by reference in their entirety.

In one aspect, disclosed method, composition, system, device and kit can be used for carrying out unmarked nucleic acid survey Sequence is based particularly on the nucleic acid sequencing of ion.The concept for the markless detection that nucleotide is incorporated to has been described in document, including such as Under document (it is incorporated herein by reference): the U.S. Patent Publication 2009/0026082 of Rothberg et al.；Anderson Et al., Sensors and Actuators B Chem., 129:79-86 (2008)；With Pourmand et al., Proc.Natl.Acad.Sci.,103:6466-6470(2006).Briefly, in nucleic acid sequencing application, it is polymerize by measurement The natural by-product of the extension of enzymatic includes hydrogen ion, polyphosphate, PPi and Pi (such as when there are pyrophosphatase) It is incorporated to measure nucleotide.The example of such method for nucleic acid sequencing and platform based on ion includes Ion Torrent PGM^TM Or Proton^TMSequenator (Ion Torrent^TM Systems,Life Technologies Corporation)。

In some embodiments, present disclosure generally relates to sequencing by teaching content provided herein The method of the nucleic acid expanded.In an exemplary embodiment, present disclosure is generally related to from polynucleotides The method for obtaining sequence information, comprising: (a) amplification of nucleic acid；(b) nucleic acid for the amplification that will be generated during the step (a) is extremely Few one is used as template to carry out template-dependent nucleic acid synthesis.Optionally according to any amplification method described herein into Row amplification.

In some embodiments, Template Dependent synthesis includes by one or more nucleotide with template dependent manner It is incorporated to newly synthesized nucleic acid chains.

Optionally, method, which may also include, generates one or more ionic byproducts that such nucleotide is incorporated to.

In some embodiments, method may also include one or more nucleotide being incorporated into sequencing primer of detection. Optionally, detection may include detecting hydrionic release.

In another embodiment, the method that present disclosure generally relates to sequencing nucleic acid, comprising: (a) basis The methods disclosed herein amplification of nucleic acid；(b) nucleic acid of amplification is placed in multiple reaction chambers, wherein the one of reaction chamber or more It is a to be contacted with field effect transistor (FET).Optionally, method further include the amplification being placed in one of reaction chamber nucleic acid with Polymerase contact, to synthesize new nucleic acid chains by the way that one or more nucleotide are continuously incorporated to nucleic acid molecules.Optionally Ground, method further include the by-product for generating one or more hydrogen ions and being incorporated to as such nucleotide.Optionally, method is also wrapped It includes by using the one or more hydrionic generations of FET detection and detects being incorporated to for one or more nucleotide.

In some embodiments, detection includes the voltage and or current response one in detection array at least one FET The variation of a or multiple hydrionic generations.

In some embodiments, FET can be selected from: ion-sensitive FET (isFET) and chemosensitivity FET (chemFET)。

It is Ion Torrent including an exemplary system by the sequencing for detecting the ionic byproducts that nucleotide is incorporated to PGM^TMOr Proton^TMSequenator (Life Technologies) is by detecting the by-product for being produced as nucleotide and being incorporated to Hydrogen ion carry out the sequencing system based on ion of sequencing nucleic acid template.Normally, hydrogen ion is released as being situated between by polymerase Existing nucleotide is incorporated to by-product during the template-dependent nucleic acid synthesis led.Ion Torrent PGM^TMOr Proton^TMIt surveys Sequence instrument is incorporated to by detecting the hydrogen ion by-product that nucleotide is incorporated to detect nucleotide.Ion Torrent PGM^TMOr Proton^TMSequenator may include multiple to be sequenced nucleic acid-templated, wherein to be placed in respective sequencing in array anti-for each template It answers in hole.The hole of array can respectively be coupled at least one ion transducer, and the sensor is detectable to be incorporated to as nucleotide By-product generate H⁺The release of ion or the variation of pH value of solution.Ion transducer includes being coupled to that H can be incuded⁺Ion is deposited Or pH value of solution variation ion-sensitive detection layers field effect transistor (FET).Ion transducer can provide instruction core The output signal that thuja acid is incorporated to, the H being represented by voltage change, the magnitude of the voltage change and respective hole or reaction chamber⁺Ion concentration is related.Different nucleotide types can be continuously flowed into reaction chamber, and can be by polymerase by the sequence of template The sequence that column determine is incorporated to extension primer (or polymerization site).Each nucleotide is incorporated to can be along with H in reacting hole⁺Ion Release and the parallel variation of local pH.H⁺The release of ion can be recorded by the FET of sensor, generate instruction and nucleotide occurs The signal being incorporated to.The nucleotide not being incorporated into specific nucleotide stream can not generate signal.The wave amplitude of signal from FET Can also be related to the extension quantity of certain types of nucleotide of nucleic acid molecules is incorporated to, to allow to parse homopolymer region.Cause This is incorporated to prison into multiple nucleotide streams of reaction chamber and in large number of orifices or reacting chamber space during the operation of sequenator It surveys and equipment is allowed simultaneously to parse many nucleic acid-templated sequences.About Ion Torrent PGM^TMOr Proton^TMSequenator Composition, design and other details of operation be found in such as U.S. Patent Application Serial 12/002781, be now disclosed as the U.S. Patent publication No. 2009/0026082；U.S. Patent Application Serial 12/474897, is now disclosed as U.S. Patent Publication No. 2010/0137143；With U.S. Patent Application Serial 12/492844, it is now disclosed as U.S. Patent Publication No. 2010/0282617 In, above-mentioned reference is incorporated herein by reference in their entirety.

Figure 28 is shown for the block diagram according to the component of the system of the nucleic acid sequencing of exemplary implementation scheme.Component packet Include the flow cell 101 on IC apparatus 100, reference electrode 108, for sequencing multiple reagents 114, valve body 116, wash Wash solution 110, valve 112, fluid control 118, route 120/122/126, channel 104/109/111, waste material container 106, battle array Column controller 124 and user interface 128.IC apparatus 100 includes the microwell array 107 covered on an array of sensors, institute Stating sensor array includes chemical sensor as described in this article.Flow cell 101 includes entrance 102, outlet 103 and is defined on The flow chamber 105 of reagent flow path on microwell array 107.

Reference electrode 108 can have any type appropriate or shape, including with flow channel or insertion channel 111 The coaxial clyinder of conducting wire in chamber.Can be driven by pump, air pressure or other methods appropriate reagent 114 by flow path, Valve and flow cell 101, and can be discharged it in waste material container 106 behind the outlet 103 for leaving flow cell 101.Fluid control Device 118 can be used software control appropriate for the operation of the driving force, valve 112 and valve body 116 of reagent 114.

The array of microwell array 107 including reaction zone as described in this article, also referred to as micropore, can operate herein Combine with chemical sensor corresponding in sensor array on ground.For example, each reaction zone can be coupled to suitable for detection reaction The chemical sensor of purpose analyte or reaction property in area.Microwell array 107 can be integrated into IC apparatus 100 In, so that microwell array 107 and sensor array are the parts of single device or chip.

Flow cell 101 can have a variety of for controlling the knot in path and flow rate of the reagent 114 on microwell array 107 Structure.Array control unit 124 is that IC apparatus 100 provides bias voltage and timing and control signal for reading sensing The chemical sensor of device array.Array control unit 124 is also provided referring to bias voltage for reference electrode 108 with to flowing through micropore battle array 114 biasing of reagent of column 107.

During the experiment, array control unit 124 is collected and chemical sensor of the processing from sensor array passes through collection Output signal at the output port on circuit device 100 Jing Guo bus 127.Array control unit 124 can be computer or its Its calculating instrument.Array control unit 124 may include memory for storing data and application software, for access data and It executes the processor of application and promotes the component with the communication of the various assemblies of the system in Figure 28.

The value instruction occur in the correspondence reaction zone in microwell array 107 one of the output signal of chemical sensor or The physically and/or chemically parameter of multiple reactions.For example, in an exemplary embodiment, can be used and disclosed in following reference Technical treatment output signal value: the U.S. Patent Application No. 13/339 that Rearick et al. was submitted on December 29th, 2011, 846, it January 3 was submitted based on the U.S. Provisional Patent Application No. 61/428,743 and 2011 year submitted on December 30th, 2010 U.S. Provisional Patent Application No. 61/429,328 and Hubbell are in the U.S. Patent Application No. submitted on December 29th, 2011 13/339,753, the U.S. Provisional Patent Application No. 61/428,097 submitted based on December 29th, 2010.

User interface 128 can be shown about flow cell 101 and received from the sensor array on IC apparatus 100 Chemical sensor output signal information.User interface 128 may also display equipment and be set and controlled and user be allowed to input Or setting equipment is set and controlled.

In an exemplary embodiment, fluid control 118 can be by individual reagent 114 to flow cell 101 during the experiment Delivering with IC apparatus 100 is controlled in predetermined order, predetermined duration, predetermined stream Speed.The chemistry that array control unit 124 then can collect and analyze the chemical reaction that instruction occurs in response to the delivering of reagent 114 passes The output signal of sensor.

During the experiment, the temperature of IC apparatus 100 can be also monitored and controlled in system, so as to known true in advance It reacts and measures at a temperature of fixed.

Configurable system is so that single fluid or reagent contact reference electrode in entire multistep reaction during operation 108.Valve 112 can be closed to prevent any washing solution 110 when reagent 114 flows in flow channel 109.Although washing solution Flowing can be prevented from, but between reference electrode 108, channel 109 and microwell array 107 can there are still continuous fluid and Telecommunication.The distance between the tie point between reference electrode 108 and channel 109 and 111 may be selected, so as to no or minimal amount of The reagent for flowing and possibly being diffused in channel 111 in channel 109 reaches reference electrode 108.In exemplary implementation scheme In, washing solution 110 can be selected as and be continuously contacted with reference electrode 108, this can be particularly used for using frequent purge step Rapid multistep reaction.

Figure 29 shows the sectional view and expanded view of the part of IC apparatus 100 and flow cell 101.During operation, The flow chamber 105 of flow cell 101 closes the examination for flowing through the reagent through delivering of open end of the reaction zone in microwell array 107 Agent stream 208.It can be selected based on the property of the reaction of generation and the labelling technique used (if any) or reagent, by-product Select the volume, shape, length-width ratio (such as bottom width is to hole depth rate) and others size characteristic of reaction zone.

Relevant reaction in chemical sensor induction (and generating output signal) microwell array 107 of sensor array 205 Chemical reaction in area is analyzed with testing goal or or reaction property.Can be for example of chemical sensor of sensor array 205 It learns sensibility field effect transistor (chemFET), such as ion-sensitive field effect transistor (ISFET).It can be used for embodiment party The chemical sensor of case and the example of array structure are described in U.S. Patent Application Publication No. 2010/0300559,2010/ 0197507,2010/0301398,2010/0300895,2010/0137143 and 2009/0026082 and U.S. Patent number 7, In 575,865, each above-mentioned reference is incorporated herein by reference.

Figure 30 is shown according to two of exemplary implementation scheme representative chemical sensors and its corresponding reaction zone Sectional view.In Figure 30, it is shown that two chemical sensors 350,351, representative may include the biography of millions of chemical sensors The sub-fraction of sensor array.In some embodiments, sensor array may include at least 1,000,000 chemical sensors and appoint Selection of land at least 1,000,000 corresponding reaction zones, at least 1,000 ten thousand chemical sensors and optionally at least 1,000 ten thousand it is corresponding anti- Answer area, at least 100,000,000 chemical sensors and optionally at least 100,000,000 corresponding reaction zones and are appointed at least 500,000,000 chemical sensors Selection of land at least 500,000,000 corresponding reaction zones or at least 1,000,000,000 chemical sensors and optionally at least 1,000,000,000 corresponding reactions Area.

Chemical sensor 350 is coupled to corresponding reaction zone 301, and chemical sensor 351 is coupled to corresponding reaction zone 302.Chemical sensor in 350 representative sensor array of chemical sensor.In the example of display, chemical sensor 350 is Ion-sensitive field effect transistor.Chemical sensor 350 includes floating gate structure 318, and the floating gate structure 318 has logical Cross the floating gate conductor (herein referred to as sensor board 320) isolated with reaction zone 301 of sensing material 316.In Figure 30 Display, the uppermost pattern layer of conductive material of the sensor board 320 in the floating gate structure 318 below reaction zone 301.

In the example of display, floating gate structure 318 includes the multiple of the conductive material in the layer of dielectric substance 319 Pattern layer.As described in greater detail below, the upper surface of sensing material 316 is used as the sensing surface of chemical sensor 350 317。

In the embodiment of display, sensing material 316 is ion-sensitive material, thus ion or other charge species Presence in the solution in reaction zone 301 changes the surface potential of sensing surface 317.The variation of surface potential is because in solution Caused by the protonation or deprotonation of surface charge group at sensing surface caused by existing ion.Sensing material 316 can be placed in by using multiple technologies, or during being used to form one or more manufacturing process of chemical sensor 350 Natural feature at.In some embodiments, sensing material 316 is metal oxide, for example, silicon, tantalum, aluminium, lanthanum, titanium, zirconium, hafnium, The oxide of tungsten, palladium, iridium etc..

In some embodiments, sensing material 316 is the oxide on the upper layer of the conductive material of sensor board 320.Example Such as, the upper layer of sensor board 320 can be titanium nitride, and sensing material 316 may include titanium oxide or titanium oxynitrides.More generally, Sensing material 316 may include the one or more of a variety of different materials to promote the sensibility to specific ion.For example, silicon nitride Or silicon oxynitride and metal oxide such as silica, aluminium oxide or tantalum oxide are usually provided to hydrionic sensibility, so It and include that the sensing material of the polyvinyl chloride with valinomycins provides the sensibility to potassium ion.Depending on instrument, can also make With the material to other ions (such as sodium, silver, iron, bromine, iodine, calcium and nitrate) sensitivity.

Chemical sensor 350 further includes source area 321 and drain region 322 in semiconductor substrate 354.321 He of source area Drain region 322 includes the semiconductor material of doping, has the conductivity type different from the conductivity type of substrate 354.Example Such as, source area 321 and drain region 322 may include the P- type of semiconductor material of doping, and substrate may include the N- type half of doping Conductor material.

Slot area 323 separates source area 321 and drain region 322.Floating gate structure 318 overlays on slot area 323, and passes through Gate-dielectric 352 is separated with substrate 354.Gate-dielectric 352 can be for example silica.Alternatively, other dielectrics can For gate-dielectric 352.

As shown in Figure 30, reaction zone 301 extends through the packing material 310 on dielectric substance 319.Packing material 310 can be for example comprising one or more layers dielectric substance such as silica or silicon nitride.

The scale (such as width and depth) of reaction zone 301,302 and its pitch (center between adjacent reaction area away from From) can between instrument and instrument it is different.In some embodiments, reaction zone can have no more than 5 microns, such as no more than 3.5 microns, it is no more than 2.0 microns, is no more than 1.6 microns, be no more than 1.0 microns, be no more than 0.8 micron, it is micro- is no more than 0.6 Rice is no more than 0.4 micron, and the characteristic diameter no more than 0.2 micron or no more than 0.1 micron, the diameter is by 4 multiplied by plan view Cross-sectional area (A) divided by Pi square root (for example, sqrt (4*A/ π)) Lai Dingyi.

In some embodiments, the pitch between adjacent reaction area is no more than 10 microns, is no more than 5 microns, is no more than 2 Micron is no more than 1 micron or no more than 0.5 micron.

In the embodiment of display, reaction zone 301,302 is separated with the distance for being equal to its width.Alternatively, adjacent anti- The separating distance between area is answered to be smaller than its width.For example, separating distance can be for being used to form reaction zone 301,302 The minimum feature size of technique (such as litho technique).In this case, separation is less than individual with directly can dramatically The width of reaction zone.

Sensor board 320, sensing material 316 and reaction zone 301 can be for example with circular cross sections.Alternatively, these can be with It is non-circular.For example, cross section can be square, it is rectangle, hexagon or irregular shape.

Depending on wherein realizing that the device and array structure of chemical sensor described herein, the device in Figure 30 may be used also Including other element for example for accessing the array line (such as wordline, bit line etc.) of chemical sensor, in substrate 354 in addition Doped region and other circuits (such as access circuit, biasing circuit etc.) for operating chemical sensor.In some embodiment party It, can be for example using U.S. Patent Application Publication No. 2010/0300559,2010/0197507,2010/0301398,2010/ in case 0300895,2010/0137143 and 2009/0026082 and U.S. Patent number 7,575,865 (each of which by reference simultaneously Enter herein) described in technology process units.

In operation, reactant, washing solution and other reagents can be moveable into and out reaction zone by flooding mechanism 340 301.The induction of chemical sensor 350 (and generating relevant output signal) is present in the sensing material opposite with sensor board 320 The amount of charge 324 on 316.The variation of charge 324 causes the voltage change on floating gate structure 318, and then causes crystal The variation of the threshold voltage of pipe.It can be measured by the electric current in the slot area 323 between measurement source area 321 and drain region 322 The variation of the threshold voltage.Therefore, chemical sensor 350, which can be used directly for providing, is connected to source area 321 or drain region 322 Array line on the output signal based on electric current, or provided indirectly using other circuit based on voltage output letter Number.

In embodiments, the reaction carried out in reaction zone 301 can be to identify or measure purpose analyte The analysis of characteristic or property is reacted.Such reaction can either directly or indirectly have an impact the charge near sensor board 320 The by-product of amount.If such by-product generate in a small amount decay rapidly or with other component reactions, can simultaneously anti- Multiple copies that same analyte is analyzed in area 301 are answered, to increase the output signal generated.In embodiments, it can will analyze Multiple copies of object are connected to solid support 312 before or after being placed in reaction zone 301.Solid support 312 can be with It is particle, nano particle, globule, solid or porous including gel etc..For simplicity and ease of explanation, solid support 312 are also referred to as particle herein.For nucleic acid analyte, the copy of multiple connections can pass through rolling circle amplification (RCA), index RCA, recombinase polymeric enzymatic amplification (RPA), polymerase chain reaction amplification (PCR), emulsion PCR amplification or similar technology are made It is standby, to generate amplicon without solid support.

In multiple exemplary implementation schemes, method, system and computer-readable medium described herein can be advantageous Ground is used to handling and/or analyzing the data and signal obtained from the nucleic acid sequencing based on electronics or charge.It is being based on electronics or charge Sequencing (for example, sequencing based on pH) in, the day of polymerase catalysed nucleotide extension can be generated as by detecting The ion (such as hydrogen ion) of right by-product measures nucleotide and incoming event.This can be used for being sequenced sample or template nucleic acid, It can be the segment of such as purpose nucleic acid sequence, and it either directly or indirectly can be connected to solid support as clone group Such as particle, particle, globule etc..Sample or template nucleic acid can operationally associate to primer and polymer and can undergo repetition Nucleotide addition and washing circulation or " stream " (herein its can be referred to " nucleotide stream ", this " nucleotide stream " can lead Nucleotide is caused to be incorporated to).Primer can be annealed to sample or template so as to whenever the core of addition and next base complementrity in template 3 ' ends of primer can be extended by polymerase when thuja acid.Sequence subsequently, based on known nucleotide stream and based on the change of measurement The output signal for learning the ion concentration during each nucleotide stream of instruction of sensor, can measure and be present in and be coupled to chemical biography Type, the information of sequence and quantity of the nucleotide of sample nucleic acid association in the reaction zone of sensor.

It, can be anti-by polymerase catalysed extension by detecting in the embodiment of the nucleic acid sequencing typically based on ion The hydrionic presence that should be generated and/or concentration are incorporated to detect nucleotide.In one embodiment, can will optionally with survey The template that sequence primer and/or polymerase combine in advance is loaded to reaction chamber and (such as is disclosed in Rothberg cited herein et al. Micropore) in, followed by duplicate nucleotide add and wash circulation.In some embodiments, such template It can be used as clone group and be connected to solid support such as particle, globule or similar, and the clone group is loaded into reaction chamber.

In another embodiment, array will be allocated in, be placed in or is positioned at optionally in combination with the template to polymerase Different loci.The site of array includes primer and method may include that different templates is hybridized to drawing in different sites Object.

In each addition step of circulation, only when the complement that next base in template is the nucleotide of addition When, polymerase can be by being incorporated to the nucleotide of addition come extension primer.If there is a complementary base, then there is one simultaneously Enter, if there are two, there are two to be incorporated to, if there are three, it is incorporated to, etc. there are three.As each It is incorporated to, there are the hydrogen ions of release, and jointly the template group of release hydrogen ions changes the local pH of reaction chamber.It is hydrionic It generates (and total with the template molecule of primer and polymer participation extension with the quantity of complementary base continuous in template Quantity) it is monotonically correlated.Therefore, when in template there are when many consecutive identical complementary base (i.e. homopolymer regions), generation Hydrionic quantity and the thus magnitude of caused local pH variation, can be proportional to the quantity of consecutive identical complementary base. If the next base and the nucleotide of addition in template be not complementary, it is incorporated to and does not occur and not release hydrogen ions.? In some embodiments, after the step of each adds nucleotide, additional step can be carried out, wherein will predefine PH under be used to remove the nucleotide of previous steps without buffering washing solution to prevent the mistake incorporation in circulation later.? In some embodiments, after the step of each adds nucleotide, additional step can be carried out, wherein being destroyed with nucleotide Reagent such as apyrase process chamber is retained in indoor any remaining nucleotide, these cores to remove Thuja acid can lead to false extension in subsequent circulation.

In an exemplary embodiment, different types of nucleotide is continuously added to reaction chamber, so as to each Reaction can be exposed to different nucleotide one at a time.For example, nucleotide: dATP, dCTP can be added by following sequence, DGTP, dTTP, dATP, dCTP, dGTP, dTTP, etc.；Washing step is carried out after exposing each time.Depending on desired sequence The length of information repeats circulation 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 750 times or more times.

It in some embodiments, can be according to PGM^TMOr Proton^TMThe user's manual that sequenator provides is sequenced. Embodiment 3 is provided for using Ion Torrent PGM^TMSequenator (Ion Torrent^TM Systems,Life Technologies, CA) the sequencing based on ion an exemplary arrangement.

In some embodiments, the method that present disclosure generally relates to sequencing template polynucleotides group, comprising: (a) multiple amplicons are generated by expanding multiple template polynucleotides with cloning on multiple surfaces, wherein mixing in reaction Carried out in the single continuous phase of object the amplification and wherein caused by amplicon at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inherently substantially monoclonal.In some embodiments, in list The amplicon of sufficient amount of substantially monoclonal is generated in a amplified reaction in Ion Torrent PGM^TM314,316 or The AQ20 sequencing that at least 100MB, 200MB, 300MB, 400MB, 500MB, 750MB, 1GB or 2GB are generated on 318 sequenators is read Number.As used herein, term " AQ20 " and its variant refer in Ion Torrent PGM^TMMeasurement sequencing is accurate in sequenator The ad hoc approach of degree.Can in a manner of Phred- sample Q score accuracy of measurement, the Phred- sample Q score is in logarithmic scale Upper accuracy of measurement: Q10=90%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50=99.999%.Example Such as, in specific sequencing reaction, standard can be calculated by prediction algorithm or by practical compare with known reference gene group Exactness measurement.The mass fraction (" Q score ") of prediction can derive from such algorithm: it considers the intrinsic property of input signal simultaneously For including whether the given single base being sequenced in " reading " will be aligned the fairly accurate evaluation of progress.In some embodiment party In case, the mass fraction of such prediction can be used for before downstream compares filtering and removal low quality reading.In some implementations In scheme, reporting accuracy, the Phred- sample Q score it can be measured in logarithmic scale in a manner of Phred- sample Q score Accuracy: Q10=90%, Q17=98%, Q20=99%, Q30=99.9%, Q40=99.99% and Q50=99.999%. In some embodiments, the data filtering of given polymeric enzyme reaction can be will be obtained from only to measure a nucleotide of measurement " N " or more Grow and have be more than the Q score of certain threshold value such as Q10, Q17, Q100 (herein referred to as " NQ17 " score) polymerase Reading.For example, 100Q20 score can indicate that be obtained from given reaction is at least 100 nucleotide in length and has Q20 (99%) or the quantity of the reading of higher Q score.Similarly, 200Q20 score can indicate to be at least 200 cores in length The quantity of thuja acid and the reading with Q20 (99%) or higher Q score.

In some embodiments, can also based on use the appropriate comparison of reference gene group sequence come accuracy in computation, Herein referred to as " original " accuracy.This is single-pass accuracy (single pass accuracy) comprising with it is single The measurement of the "true" of relevant every base mistake is read, this is with shared accuracy on the contrary, shared accuracy measurement comes from shared sequence The error rate of column is the result of multiple readings.Original can be reported in a manner of " AQ " (writing a Chinese character in simplified form for " quality of comparison ") score Beginning accuracy measurement.In some embodiments, the data filtering of given polymeric enzyme reaction can be will be obtained from only to measure measurement " N " A nucleotide or it is longer and have be more than certain threshold value such as AQ10, AQ17, AQ100 (herein referred to as " NAQ17 " divide Number) AQ score polymerase reading.For example, 100AQ20 score can indicate to be obtained from given polymeric enzyme reaction be in length The quantity of at least 100 nucleotide and the reading with AQ20 (99%) or higher AQ score.Similarly, 200AQ20 points Number can indicate be in length at least 200 nucleotide and the reading with AQ20 (99%) or higher AQ score number Amount.

In some embodiments, this teaching content provides the system for nucleic acid amplification, and the system includes following Any combination: be connected with globule, the second primer, third primer, polynucleotides, the recombinase, recombinase of multiple the first primers It is loaded into albumen, single strand binding protein (SSB), polymerase, nucleotide, ATP, phosphocreatine, creatine kinase, hybridization solution and/or washes Wash solution.System may include all or some of these components.In some embodiments, may be used also for the system of nucleic acid amplification Any combination comprising buffer and/or cation (such as bivalent cation).

In some embodiments, this teaching content provides the kit for nucleic acid amplification.In some embodiments In, kit includes any reagent that can be used for nucleic acid amplification.In some embodiments, kit may include following any Combination: globule, the second primer, third primer, polynucleotides, recombinase, the recombinase for being connected with multiple the first primers are loaded into egg White, single strand binding protein (SSB), polymerase, nucleotide, ATP, phosphocreatine, creatine kinase, hybridization solution, washing solution, buffering Liquid and/or cation (such as bivalent cation).Kit may include all or some of these components.

In some embodiments, present disclosure generally relates in multiple divided reaction capacities in parallel Expand different nucleic acid-templated methods (such as opposite with the amplification in single Continuous Liquid Phase), composition, system.For example, Can by it is nucleic acid-templated distribution to or be placed in the array of reaction chamber or the array of reaction capacity, so as at least two in array this The room of sample or capacity respectively receive single nucleic acid-templated.In some embodiments, multiple isolated reaction capacities are formd.It can Closed reaction chamber's (or reaction capacity) optionally before amplification.In another embodiment, reaction mixture can be divided Or separation is into multiple microreactors in the continuous phase for being dispersed in emulsion.Divided or isolated reaction capacity optionally not that This mixing or connection, or can not be mixed with each other or be connected to.In some embodiments, (or reaction is held at least some reaction chambers Amount) include recombinase and optionally polymerase.Polymerase can be strand displacement polymerase.

In some embodiments, present disclosure generally relates to nucleic acid synthesis and/or what is expanded includes emulsion Composition, system, method, apparatus and kit.As used herein, term " emulsion " includes comprising the first liquid and the second liquid Any composition of the mixture of body, wherein the first and second liquid are substantially immiscible each other.Normally, the one of liquid It is hydrophobic that kind, which is hydrophilic and other liquid,.Normally, emulsion includes dispersed phase and continuous phase.For example, the first liquid Body can form the dispersed phase being dispersed in second liquid, and the second liquid forms continuous phase.Dispersed phase is optionally mainly by One liquid composition.Continuous phase is optionally mainly made of second liquid.In various embodiments, identical two kinds of liquid can shape At different types of emulsion.For example, the mixture comprising oil and water can be initially formed oil-in-water emulsion, wherein oil is dispersion Phase, water are decentralized media.Secondly, they can form water-in-oil emulsion, wherein water is dispersed phase, and oil is foreign minister.Multiple emulsion And it is possible, including " W/O/W " emulsion and " Water-In-Oil packet oil " emulsion.In some embodiments, dispersed phase includes One or more wherein nucleic acid-templated microreactors that can be amplified eachly.One or more microreactors, which can be formed, wherein may be used The divided reaction capacity of isolated amplified reaction.One example of the medium appropriate for nucleic acid amplification includes Water-in-oil emulsion, wherein based on water mutually include several aqueous microreactors being dispersed in the oily phase of emulsion.In some realities It applies in scheme, emulsion also may include emulsifier or surfactant.Emulsifier or surfactant can be used for synthesizing item in nucleic acid Stable emulsion under part.

In some embodiments, present disclosure is usually directed to the composition comprising emulsion, and the emulsion includes reaction Mixture.Emulsion may include water phase.Water phase is dispersed in the continuous phase of emulsion.Water phase may include one or more micro- reactions Device.In some embodiments, reaction mixture includes in multiple liquid phase microreactors in the phase of emulsion.Optionally, instead Answering mixture includes recombinase.Optionally, reaction mixture includes multiple and different polynucleotides.Optionally, reaction mixture Include multiple supports.Optionally, reaction mixture includes recombinase, multiple and different polynucleotides and/or multiple supports Any combination.Optionally, at least one support can be connected to the nucleic acid group of substantially monoclonal.

In some embodiments, present disclosure is usually directed to the composition comprising reaction mixture, and the reaction is mixed Closing object includes (i) multiple supports, (ii) multiple and different polynucleotides and (iii) recombinase, and reaction mixture is included in cream In multiple liquid phase microreactors in agent.

In some embodiments, present disclosure is usually directed to the composition comprising reaction mixture, and the reaction is mixed Closing object includes (i) recombinase and (ii) multiple supports, at least one support can be connected to the nucleic acid group of substantially monoclonal, Wherein reaction mixture includes in multiple liquid phase microreactors in emulsion.

Optionally, emulsion includes aqueous favoring.

Optionally, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.For example, emulsion may include water-in-oil emulsion.

In some embodiments, aqueous favoring includes multiple microreactors.

Optionally, reaction mixture is included in single reaction vessel.

Optionally, the sequence of multiple and different polynucleotides can be identical or different.

Optionally, at least one of multiple supports is connected to multiple the first primers (such as positive amplimer).

Optionally, reaction mixture also includes multiple second primers (such as reversed amplimer).

In some embodiments, at least one of multiple supports also includes multiple second primers.

In some embodiments, at least one of multiple supports includes multiple first and second primers.

In some embodiments, the first and second primers include identical sequence.In some embodiments, the first He Second primer includes different sequence.

In some embodiments, support includes the inner wall of globule, particle, plane surface or slot or pipe.

In some embodiments, reaction mixture also includes polymerase and multiple nucleotide.

In some embodiments, present disclosure is usually directed to the composition comprising emulsion.Optionally, emulsion includes parent Water phase and hydrophobic phase.Optionally, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.Optionally, aqueous favoring may include multiple more Any combination of oligonucleotide template, multiple supports and/or recombinase.Optionally, aqueous favoring may include multiple polynucleotides moulds Plate.Optionally, aqueous favoring may include multiple supports.Selection of land, aqueous favoring may include recombinase.

In some embodiments, composition includes emulsion, and the emulsion includes aqueous favoring and hydrophobic phase, wherein aqueous favoring Include multiple polynucleotide templates, multiple supports and recombinase.

In some embodiments, present disclosure is usually directed to the composition comprising emulsion, and the emulsion includes dispersion Aqueous favoring in hydrophobic phase.Optionally, aqueous favoring includes multiple microreactors.Optionally, at least two in multiple are micro- anti- Answering device includes different polynucleotide templates.Optionally, the sequence of different polynucleotide templates is identical or different.Optionally Ground, the first microreactor includes the first polynucleotide template and the second microreactor includes the second polynucleotide template.Optionally Ground, the first and second polynucleotide templates include identical or different sequence.Optionally, at least two microreactors in multiple Include recombinase.

In some embodiments, composition includes emulsion, and the emulsion includes the aqueous favoring being dispersed in hydrophobic phase, Middle aqueous favoring include multiple microreactors, it is multiple at least two microreactors include different polynucleotide templates and recombination Enzyme.

In some embodiments, aqueous favoring includes multiple aqueous microreactors, and at least two of microreactor respectively wrap Containing different polynucleotide templates, support and recombinase.

Optionally, the first microreactor includes the first polynucleotide template and the second microreactor includes the second multicore glycosides Acid template.Optionally, the first and second polynucleotide templates include identical or different sequence.

In some embodiments, the first and second primers include identical sequence.

In some embodiments, the first and second primers include different sequences.In some embodiments, aqueous favoring It also include polymerase.

In some embodiments, polymerase includes strand displacement polymerase.In some embodiments, aqueous favoring includes core Thuja acid.

In some embodiments, present disclosure generally relates to nucleic acid synthetic method (and relevant composition And system), comprising: (a) forms reaction mixture；(b) reaction mixture is made to undergo amplification condition.Optionally, reaction mixing Object is included in the aqueous favoring of emulsion.Optionally, emulsion includes aqueous favoring and hydrophobic phase.Optionally, emulsion is thin comprising being dispersed in Aqueous favoring in water phase.Optionally, reaction mixture includes multiple supports, multiple and different polynucleotides and/or recombinase Any combination.Optionally, reaction mixture includes multiple supports.Optionally, reaction mixture includes multiple and different more Nucleotide.Optionally, the sequence of different polynucleotide templates is identical or different.Optionally, the first microreactor includes First polynucleotide template and the second microreactor include the second polynucleotide template.Optionally, the first and second multicore glycosides Acid template includes identical or different sequence.Optionally, reaction mixture includes recombinase.Optionally, amplification condition include etc. Temperature or thermal cycling temperature condition.Optionally, method further includes forming at least two supports to lead to emulsion experience amplification condition Multiple supports are formed, wherein at least two of support are connected to the nucleic acid group of substantially monoclonal each independently.

In some embodiments, present disclosure generally relates to nucleic acid synthetic method (and relevant composition And system), comprising: (a) forms the reaction mixture comprising multiple supports, multiple and different polynucleotides and recombinase, instead Mixture is answered to be included in the aqueous favoring of emulsion；(b) make the emulsion experience isothermal duplication condition comprising reaction mixture, thus It generates multiple supports and is connected to the nucleic acid group of its substantially monoclonal.

In some embodiments, emulsion includes water-in-oil emulsion.In some embodiments, liquid phase microreactor packet Containing aqueous favoring.In some embodiments, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.In some embodiments, exist Reaction mixture is formed in single reaction vessel.Optionally, the sequence of multiple and different polynucleotide templates is identical or different 's.Optionally, the first polynucleotide template includes First ray and the second polynucleotide template includes the second sequence.Optionally Ground, the first and second polynucleotide template sequences are identical or different.Optionally, at least one of multiple supports is connected to Multiple the first primers (such as positive amplimer).Optionally, reaction mixture also includes that multiple second primers (such as reversed expand Increase primer).In some embodiments, at least one of multiple supports also includes multiple second primers.In some embodiment party In case, at least one of multiple supports includes multiple first and second primers.In some embodiments, first and second draw Object includes identical sequence.In some embodiments, the first and second primers include different sequences.In some embodiments In, nucleic acid synthesis methods further include that at least some supports for being connected to substantially nucleic acid monoclonal group are recycled from reaction mixture Object.In some embodiments, nucleic acid synthesis methods further include by least some nucleic acid groups for being connected to substantially monoclonal Support is placed on the surface.In some embodiments, nucleic acid synthesis methods further include by being connected to base at least some The support of the nucleic acid group of monoclonal is placed on surface to form array in sheet.In some embodiments, nucleic acid synthesis side Method further includes that at least one is sequenced to be connected to the nucleic acid group of the substantially monoclonal of support.In some embodiments, it supports Object includes the inner wall of globule, particle, plane surface or slot or pipe.In some embodiments, reaction mixture also includes polymerization Enzyme and multiple nucleotide.In some embodiments, polymerase includes strand displacement polymerase.

It in some embodiments, include forming emulsion for nucleic acid synthetic method.Optionally, emulsion includes aqueous favoring And hydrophobic phase.Optionally, emulsion includes the aqueous favoring being dispersed in hydrophobic phase.Optionally, aqueous favoring includes multiple microreactors. Optionally, at least two microreactors in multiple include individual polynucleotide template.Optionally, at least two in multiple are micro- Reactor includes different polynucleotide template.Optionally, the first microreactor includes the first polynucleotide template, and second Microreactor includes the second polynucleotide template.Optionally, the first and second polynucleotide templates have identical or different sequence Column.Optionally, at least two microreactors in multiple include recombinase.

In some embodiments, present disclosure generally relates to nucleic acid synthetic method (and relevant composition And system), comprising: the emulsion of the aqueous favoring comprising being dispersed in hydrophobic phase is formed, the aqueous favoring includes multiple microreactors, At least two microreactors in multiple include different polynucleotide template and recombinase.

In some embodiments, emulsion includes water-in-oil emulsion.In some embodiments, aqueous favoring also includes poly- Synthase.In some embodiments, polymerase is strand displacement polymerase.In some embodiments, aqueous favoring includes nucleotide. In some embodiments, emulsion is formed in single reaction vessel.Optionally, the sequence of different polynucleotide templates is phase It is same or different.Optionally, the first microreactor includes the first polynucleotide template and the second microreactor includes more than second Oligonucleotide template.Optionally, the first and second polynucleotide templates include identical or different sequence.In some embodiments In, it is multiple at least two microreactors include multiple supports.Optionally, at least one of multiple supports is connected to more A the first primer (such as positive amplimer).Optionally, reaction mixture also includes (such as the reversed amplification of multiple second primers Primer).In some embodiments, at least one of multiple supports also includes multiple second primers.In some embodiments In, at least one of multiple supports includes multiple first and second primers.In some embodiments, the first and second primer Include identical sequence.In some embodiments, the first and second primers include different sequences.In some embodiments In, aqueous favoring includes reaction mixture.In some embodiments, reaction mixture includes multiple polynucleotide templates, multiple Support and recombinase.It in some embodiments, further include keeping emulsion (such as mixed comprising reacting for nucleic acid synthetic method Close object) experience isothermal duplication condition, to generate the nucleic acid group of multiple substantially monoclonals.In some embodiments, multiple The nucleic acid group of substantially monoclonal is connected to multiple supports.In some embodiments, nucleic acid synthesis methods further include from anti- Mixture is answered to recycle at least some supports for being connected to substantially nucleic acid monoclonal group.In some embodiments, nucleic acid closes It further include placing the support of at least some nucleic acid groups for being connected to substantially monoclonal on the surface at method.In some realities It applies in scheme, nucleic acid synthesis methods further include by putting the support of at least some nucleic acid groups for being connected to substantially monoclonal It sets and forms array on the surface.In some embodiments, nucleic acid synthesis methods further include that at least one is sequenced to be connected to branch Hold the nucleic acid group of the substantially monoclonal of object.In some embodiments, support includes globule, particle, plane surface or slot Or the inner wall of pipe.In some embodiments, reaction mixture also includes polymerase and multiple nucleotide.In some embodiments In, polymerase includes strand displacement polymerase.

Paragraph heading used herein, which is only used for organizational goal not, should be construed as limitation description Theme.

All documents and similar material quoted in the application, including but not limited to patent, patent application, article, book Nationality, paper and internet page are integrally incorporated herein explicitly by reference for any purpose.When the reference being incorporated to In the definition of term when seeming different from the definition provided in this teaching content, should be determined with what is provided in this teaching content Subject to justice.

It will be understood that there is implicit " about " before the temperature discussed in this teaching content, concentration, number etc., therefore slight And unsubstantiality difference in the range of this teaching content.

Unless the context otherwise requires, the term comprising plural number and plural number will be included odd number by singular term.

Term "comprising", " comprising ", " containing ", " having ", " having " and " having " are not intended to be restrictive.

It will be understood that foregoing general description and detailed description below are only exemplary and explanatory and do not limit The present invention.

Unless otherwise defined, the scientific and technical terms relatively used with described herein teaching content will have By the normally understood meaning of those skilled in the art.Normally, with cell and tissue culture, molecular biology and albumen and The nomenclature and its technology that widow-or more-nucleotide chemistry and hybridization relatively use are known in the art and usually used. Standard technique is for example for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid and oligonucleotide synthesis.According to saying for manufacturer Bright book carries out as usually realized in this field or as described in this article enzymatic reaction and purification technique.Generally according to ability Well known conventional method and such as quote in entire this specification and discuss various general and referring more particularly to data in domain Described in carry out technology and methods described herein.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual(Third ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.2000).Nomenclature used in connection with and laboratory method described herein and technology are public in this field Know and usually used.

As according to used in exemplary implementation scheme provided herein, following term should be managed except as otherwise indicating Solution is with following meaning:

As used herein, term " amplification " and its variant include for generating the more of at least certain a part of polynucleotides Any process of a copy or complement, the polynucleotides are commonly referred to as " template ".Template polynucleotide can be single-stranded Or double-strand.It can lead to the generation of polynucleotide amplification product group, the polynucleotide amplification product group to the amplification of solid plate Collectively referred to as " amplicon ".The polynucleotides of amplicon can be single-stranded or double-stranded or two kinds mixtures.Normally, mould Plate will include target sequence, and generated amplicon will be comprising with sequence that is substantially the same with target sequence or being substantially complementary The polynucleotides of column.In some embodiments, the polynucleotides of specific amplicon are substantially the same or substantially mutual each other It mends；Alternatively, in some embodiments, the polynucleotides in given amplicon can have nucleotide sequence different from each other. Amplification can be carried out in a manner of linear or index, and may include to the repetition of solid plate and continuous duplication to form two A or more amplified production.Some typical amplified reactions include the continuous and repetitive cycling of the nucleic acid synthesis based on template, Lead to the formation of multiple sub- polynucleotides, the sub- polynucleotides include at least certain a part of the nucleotide sequence of template and The nucleotide sequence homology (or complementary) of at least a certain degree is enjoyed with template.In some embodiments, each core Acid synthesis (its " circulation " that can be referred to amplification) includes primer annealing and primer extension procedures；Optionally, may also include wherein The other denaturing step that template is partially or completely denaturalized.In some embodiments, an amplification bout includes single expands Increase the given number of repetition of circulation.For example, amplification bout may include particular cycle 5,10,15,20,25,30,35,40, 50,75,100 or more repetitions.In an exemplary embodiment, amplification includes wherein specific polynucleotide template experience Any reaction of two continuous nucleic acid synthesis circulations.Synthesis may include template-dependent nucleic acid synthesis.Nucleic acid synthesizes each A circulation optionally includes single primer annealing step and single extension step.In some embodiments, amplification includes isothermal Amplification.

As used herein, term " contact " and its variant, when for any group of component in use, include wherein will be to The component contacted mixes into identical mixture any process of (for example, being added into identical compartment or solution), And it is not necessarily required to that the actual physics between the component contact.It can in any order or any combination (or sub-portfolio) connects The component is touched, and may include such situation: it is wherein then by one of the component or some from mixture removal, appoint Selection of land is before the other components of addition.For example, " contacting A with B and C " includes arbitrary and whole following state: (i) A is mixed with C, B is then added to mixture；(ii) A and B are mixed into mixture；B is removed from mixture, and C is then added to mixture；A is added to the mixture of B and C by (iii)." contacting template with reaction mixture " includes Arbitrary or whole following state: (i) contacts template to generate mixture with the first component of reaction mixture；Then will Other components of reaction mixture are in any order or combination is added to mixture；(ii) reacts before mixing with template Mixture is formed completely.

As used herein, term " support " and its variant include on it can fixating reagent such as nucleic acid it is any solid Body or semisolid article.

As used herein, term " isothermal " and its variant are used when about any reaction condition of finger, process or method When, including the condition, process and method carried out under conditions of substantially isothermal.Substantially the condition of isothermal includes wherein temperature Any condition being confined in limited range.In an exemplary embodiment, the variation of temperature is no more than 20 DEG C, usually not more than Cross 10 DEG C, 5 DEG C or 2 DEG C.Isothermal duplication includes wherein carrying out at least two continuous nucleic acid under conditions of substantially isothermal to close At any amplified reaction of circulation, and including the wherein temperature during the duration of at least two continuous nucleic acid synthesis circulations The variation of degree is no more than 20 DEG C, 10 DEG C, 5 DEG C or 2 DEG C of amplified reaction, although wherein during the other parts of amplification procedure, Including during other nucleic acid synthesis circulation, the variation of temperature can be more than 20 DEG C.Optionally, in isothermal reaction (including isothermal Amplification) in, by temperature or about 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C or 70 DEG C keep at least about 10,15,20,30,45,60 or 120 Minute.Optionally, during one or more amplification cycles (for example, 1,5,10, the 20 or all amplification cycles carried out), Any temperature variation is no more than 20 DEG C, optionally within 10 DEG C, such as within 5 DEG C or 2 DEG C.In some embodiments, Isothermal duplication may include thermal cycle, and wherein temperature change is within the scope of isothermal.In instances, denaturing step and another step Such as the temperature change between annealing and/or extension is restricted.In instances, for one or more amplification cycles, denaturation Difference between temperature and annealing or elongating temperature is no more than 20 DEG C, optionally within 10 DEG C, such as within 5 DEG C or 2 DEG C. Optionally, temperature change is limited at least 5,10,15,20,30, the 35 of amplification or substantially all circulation.

As used herein, term " sequencing " and its variant include commonly by least some in measurement nucleic acid molecules The information of nucleotide (including its nucleobase component) obtains sequence information from nucleic acid chains.And in some embodiments, it " surveys The given area of sequence " nucleic acid molecules includes each nucleotide in the region that identification is sequenced, and " sequencing " may also include wherein The information of one or more nucleotide is determined and the information of some nucleotide is still undetermined or improperly determines Method.

As used herein, term " identity " and " identical " and its variant, when for two or more nucleic acid sequences Column are in use, refer to the sequence similarity of two or more sequences (such as nucleotide or polypeptide sequence).At two or more Under the background of a homologous sequence, the percentage identity or homology of sequence or its subsequence indicate all identical monomeric units (such as nucleotide or amino acid) ratio (i.e. about 70% identity, preferably 75%, 80%, 85%, 90%, 95% or 99% identity).When compare and compare on comparison window or specified region such as by using with described below default BLAST the or BLAST2.0 sequence comparison algorithm of parameter or the maximum correspondence measured by manually comparing and estimating detection When, percentage identity can be the percentage identity on specific region.When in amino acid levels or on nucleotide level In the presence of at least 85% identity when, then it is assumed that sequence is " substantially the same ".Be preferably, at least about 25 in length, On the region of 50 or 100 residues, or in the whole length of at least one sequence compared there are identity.For measuring The typical algorithm of Percent sequence identity and sequence similarity is BLAST and BLAST2.0 algorithm, is described in Altschul Et al., in Nuc.Acids Res.25:3389-3402 (1977).Other methods include Smith&Waterman, The algorithm of Adv.Appl.Math.2:482 (1981) and Needleman&Wunsch, J.Mol.Biol.48:443 (1970) etc.. Two nucleic acid sequences be another substantially the same instruction be two molecules or its complement under stringent hybridization conditions each other Hybridization.

As herein with respect to used in two or more polynucleotides, term " complementary " and its variant, which refer to, includes Accumulation base pairing can be undergone (such as in the double of hybridization on antiparallel direction in two or more individual corresponding sites In serobila) any nucleic acid sequence polynucleotides.Optionally, it may be present between the first and second nucleic acid sequences " completely " or " entire " complementary, wherein each nucleotide in the first nucleic acid sequence can be corresponding in second nucleotide sequence Polynucleotides undergo stable base pairing to interact (however, term " complementary " may include in itself in antiparallel position Non-fully complementary nucleic acid sequence over the entire length)；" part " complementary describe its more control sequences at least 20% but The nucleic acid sequence complementary with the residue in other nucleic acid sequences less than 100% residue.In some embodiments, nucleic acid sequence At least 50% but less than 100% residue it is complementary with the residue in other nucleic acid sequences.In some embodiments, nucleic acid sequence Column at least 70%, 80%, 90% or 95% but less than 100% residue it is complementary with the residue in other nucleic acid sequences.When one At least the 85% of the residue of a nucleic acid sequence and the residue mutual added time in other nucleic acid sequences, then it is assumed that sequence is " substantially mutually It mends "." incomplementarity " describes the complementary with the residue in other nucleic acid sequences less than 20% residue of its more control sequences Nucleic acid sequence.It is not at complementary any position that " mispairing ", which is contained therein two opposite nucleotide,.Complementary nucleotide packet Include the nucleotide being effectively incorporated to relative to one another during DNA replication dna by archaeal dna polymerase in physiological conditions.In typical case Embodiment in, complementary nucleotide can form base-pair each other, such as pass through the core on parallel position reversely with each other A-T/U the and G-C alkali that the hydrogen of specific Watson-Crick type between thuja acid and/or the nucleobase of polynucleotides bonds together to form Base pair.The complementary of other artificial bases couple can hydrophobic phase and/or base based on the bonding of other types of hydrogen and/or base Between shape complementary.

As herein with respect to used in any polynucleotides or nucleic acid molecules, term " double-strand " and its variant refer to tool There is one or more chain and including the region comprising the nucleotide residue with nucleotide residue base pairing (such as such as nucleic acid double chain In body) any polynucleotides or nucleic acid molecules.Optionally, the polynucleotides (or nucleic acid molecules) of double-strand can be " completely Ground " or " entirely " double-strand, in this way, each nucleotide residue in polynucleotides (or nucleic acid molecules) is corresponding with another Nucleotide residue base pairing.In some embodiments, double-stranded polynucleotide include it is one or more comprising not with it is any its The single stranded zone of the nucleotide residue of its nucleotide residue base pairing.In some embodiments, double-stranded polynucleotide (or nucleic acid Molecule) in the nucleotide residue of at least 51%, 75%, 85%, 95%, 97% or 99% match with other nucleotide residue bases It is right.In some embodiments, double-stranded polynucleotide (or nucleic acid molecules) includes two chains not being covalently attached each other；Alternatively, Double-stranded polynucleotide (or nucleic acid molecules) includes with own bases pairing at least certain a part of its length (such as such as hair clip In oligonucleotides) it is single-stranded.At least 85% when the nucleotide residue of polynucleotides matches with corresponding nucleotide residue base Clock synchronization, then it is assumed that polynucleotides are " substantially double-strands ".When a nucleic acid sequence residue with it is right in another nucleic acid sequence When the residue base pairing answered, then it is assumed that the two nucleic acid sequences are " double-strands ".In some embodiments, base pairing can Occurred according to a certain conventional pairing pattern, such as by parallel nucleotide reversely with each other and/or polynucleotides position Nucleobase between specific Watson-Crick type A-T/U the and G-C base-pair that bonds together to form of hydrogen；In other embodiment party In case, base pairing can be occurred by any other pattern, and wherein base pairing is according to established and predictable rule Come carry out.

As used herein, term " single-stranded " and its variant, when for any polynucleotides or nucleic acid molecules in use, Refer to including comprising any polynucleotides or nucleic acid not with the region of the nucleotide residue of any nucleotide residue base pairing Molecule.Optionally, single-stranded polynucleotides (or nucleic acid molecules) can be " fully " or " entirely " single-stranded, in this way, more Each nucleotide residue in nucleotide (or nucleic acid molecules) not with any other nucleotide residue base pairing.In some realities It applies in scheme, single stranded polynucleotide includes one or more double-strands comprising with the nucleotide residue of nucleotide residue base pairing Area.In some embodiments, in single stranded polynucleotide (or nucleic acid molecules) at least 51%, 75%, 85%, 95%, 97% Or 99% nucleotide residue not with other nucleotide residue base pairings.When at least the 85% of the nucleotide residue of polynucleotides When not with nucleotide residue base pairing, then it is assumed that polynucleotides are " substantially single-stranded ".

As used herein, term " denaturation " and its variant, when for any double-stranded polynucleotide molecule or double-strand multicore Nucleotide sequence including the base pairing between the nucleotide in the wherein opposite chain of duplex molecule or double-stranded sequence in use, broken Bad any process.Normally, denaturation includes at least certain a part so that double-stranded polynucleotide molecule or two chains of sequence Or region becomes single-stranded.In some embodiments, denaturation includes two chains by double-stranded polynucleotide molecule or sequence At least certain a part or region are separated from each other.Normally, the region of denaturation or part are then able to be hybridized to another multicore glycosides Acid molecule or sequence.Optionally, " complete " or " entirely " denaturation of double-stranded polynucleotide molecule or sequence may be present.Completely Denaturing is for example to will lead to the signal portions (such as more than 10%, 20%, 30%, 40% or 50%) of a large amount of chains to prolong with it The condition that complement stretch and/or overall length is kept completely separate.Normally, complete or entire denaturation destroys the nucleotide of two chains All base pairings to each other.Similarly, optionally any double when lacking more than 80% or 90% for the individual molecular of sample When chain (or lacking any hybridization to complementary strand), then it is assumed that sample of nucleic acid is denaturalized completely.

Alternatively, double-stranded polynucleotide molecule or sequence can be part or be non-fully denaturalized.When at least one of nucleic acid The part of chain keeps hybridizing with complementary strand, and another part in non-hybridized state (even if complementary strand there are the case where Under) when, it is believed that given nucleic acid molecules are partial denaturations.Non-hybridized part optionally has at least 5,7,8,10,12, 15, the length of 17,20 or 50 nucleotide.The part of hybridization optionally has at least 5,7,8,10,12,15,17,20 or 50 The length of nucleotide.Partial denaturation includes the wherein some but not all and double-stranded polynucleotide of a chain or the nucleotide of sequence The situation of some nucleotide bases pairing of interior other chains or sequence.In some embodiments, the multicore glycosides of partial denaturation The nucleotide residue of one chain of sour (or sequence) at least 20% but less than 100% not with the nucleotide residue alkali in opposite chain Basigamy pair.Under exemplary condition, the nucleotide residue in double-stranded polynucleotide molecule (or double-stranded polynucleotide sequence) is extremely Few 50% is single-stranded (or non-hybridized) form, but the residue less than 20% or 10% is double-strand.

Optionally, when the substantial portions of the individual nucleic acid molecules of sample (for example, more than 20%, 30%, 50% or 70%) when being in partial hybridization state, it is believed that sample of nucleic acid is partial denaturation.Optionally, substantial amount is less than in sample Individual nucleic acid molecules be denaturalized completely, such as the nucleic acid point in sample no more than 5%, 10%, 20%, 30% or 50% Son.Under exemplary condition, at least the 50% of the nucleic acid molecule of sample is partial denaturation, but has been less than 20% or 10% It is denaturalized entirely.In other situations, at least the 30% of the nucleic acid molecule of sample is partial denaturation, but is less than 10% or 5% It is denaturalized completely.Similarly, when individual nucleic acid molecules a small number of in sample be partially or completely be denaturalized when, it is believed that nucleic acid Sample is non denatured.

In embodiments, part Denaturing is realized by the way that duplex is maintained at temperature range appropriate.For example, Nucleic acid is maintained at and is sufficiently increased to realize some thermal denaturations (such as higher than 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C or 70 DEG C) But it is not high enough to realize complete thermal denaturation (for example, lower than 95 DEG C or 90 DEG C or 85 DEG C or 80 DEG C or 75 DEG C)

Temperature.In embodiments, using the condition part denaturing nucleic acid of substantially isothermal.

Can also by other methods, such as using concentration appropriate adjustment chemical denaturant such as urea or formamide or make With the chemical method of high or low pH (such as pH between 4-6 or 8-9), Lai Shixian partial denaturation.In embodiments, using weight Group enzyme-polymeric enzymatic amplification (RPA)

To realize partial denaturation and amplification.Exemplary RPA method description is in this article.

In some embodiments, double-stranded polynucleotide sequence to be denatured is handled by using denaturant appropriate Realize denaturation completely or partially.For example, double-strand can be made by raising the temperature to the point for the denaturation for wherein realizing aspiration level Polynucleotides undergo thermal denaturation (also by interchangeably referenced as heat denatured).In some embodiments, double-stranded polynucleotide Complete thermal denaturation, temperature-adjustable is kept completely separate with two chains for realizing polynucleotides, in this way, at least the 90% of chain is whole at its It is single stranded form in a length.In some embodiments, by being exposed to polynucleotide molecule (or sequence) higher than more Nucleic acid molecule or sequence be computed or prediction at least 5 DEG C of melting temperature (Tm), 10 DEG C, 15 DEG C, 20 DEG C, 25,30 DEG C, 50 DEG C or 100 DEG C of temperature realizes the complete thermal denaturation of polynucleotide molecule (or polynucleotide sequence).

Alternatively, chemical change can be realized by contacting double-stranded polynucleotide to be denatured with chemical denaturant appropriate Property, the chemical denaturant is, for example, highly basic, strong acid, chaotropic agent etc. and may include such as NaOH, urea or the chemical combination containing guanidine Object.In some embodiments, by being exposed to chemical denaturant such as urea or formamide or the use of concentration appropriate adjustment High or low pH (such as pH between 4-6 or 8-9) Lai Shixian is partially or completely denaturalized.In embodiments, poly- using recombinase- Synthase expands (RPA) Lai Shixian partial denaturation and amplification.Exemplary RPA method description is in this article.

When about given polynucleotides (or given target sequence in polynucleotides) in use, term " melting temperature ", " Tm " or " T_m" and its variant typically refer to such temperature, at such a temperature under determining condition group give polynucleotides The 50% of (or given target sequence) is with double-stranded form presence and 50% is single-stranded.In some embodiments, it determines Condition group may include indicating the parameter of the determination of aqueous reaction condition ionic strength and/or pH.Determining condition can be by changing Become the concentration, temperature, pH, buffer and/or formamide of salt (such as sodium) to adjust.Normally, the thermal melting point being computed It can be T_mUnder about 5-30 DEG C or T_mUnder about 5-25 DEG C or T_mUnder about 5-20 DEG C or T_mUnder about 5-15 DEG C or T_mUnder about 5-10℃.For calculating T_mMethod be well known and be found in Sambrook (1989in " Molecular Cloning:A Laboratory Manual”,2^ndedition,volumes 1-3；Wetmur 1966,J.Mol.Biol.,31:349-370； Wetmur 1991Critical Reviews in Biochemistry and Molecular Biology,26:227-259) In.It is used to hybridize or the T of denaturing nucleic acid for calculating_mOther sources include OligoAnalyze (from Integrated DNA Technologies) and Primer3 (by the Whitehead Institute for Biomedical Research Publication).In some embodiments, term " melting temperature ",

" Tm " and " T_m" and its variant include the practical Tm of given polynucleotides (or target sequence) (as using determining item What part empirically measured) or Tm that is predicting or being computed.In some embodiments, can include by assuming template The certain proportion of 4 kinds of common nucleotide acid (A, C, G and T) simultaneously has certain length (or in the case where template group, average length) Carry out the Tm of prediction or calculation template in the case where not using the sequence of template.For example, it may be assumed that being migrated to hangover on gel Template group include each 25% A, C, G or T, and with 200,300,400 base-pairs average length.

As herein with respect to used in chemical part, term " label " and its variant include comprising optically or non-optical Any composition of upper detectable part, wherein detectable part has passed through chemical treatment artificially addition, connection or attached To not labeled second part.Normally, user (or supplied upstream person) is in order to enhance the detectability of second part The addition that is marked of purpose.It is present in the optically or non-optical of the composition in the naturally occurring form of composition Upper detectable component is (for example, be present in the hydrogen ion in typical DNA molecular, RNA molecule or nucleotide in n cell And amino acid) be not intended to present disclosure purpose label.Some typical labels include fluorescence part and dyestuff.

If nucleic acid during the condition selected (such as during amplified reaction) for substantially stable mode at least to connect It is connected to support, then it is believed that nucleic acid is fixed.Any mechanism, including but not limited to non-covalent bonding, ion phase can be passed through Interaction, covalent linkage, to be attached.If the first nucleic acid is hybridized to fixed the second nucleic acid on the support, if expanded Increasing condition is so so that any or all time during amplification of the first and second nucleic acid of substantial amount is each other Association or connection, then it is also contemplated that the first nucleic acid is affixed to support during amplification.Such as first and second nucleic acid It can be by including that the hybridization of Watson-Crick base pairing or hydrogen bonding is associated together.In instances, the amplification item of selection Part allows the holding of at least 50%, 80%, 90%, 95% or 99% of the first nucleic acid and hybridizing for the second nucleic acid, or vice versa. If nucleic acid does not either directly or indirectly connect or associates with support, it is believed that nucleic acid is loose or revocable.

If medium is temporarily, at least liquid mediums object under selected conditions, the liquid mediums object is not substantive Ground or the transfer or movement for fully inhibiting or hindering loose molecule, then it is believed that medium is can under selected conditions Flowing.The molecular association that loose molecule itself is not fixed to solid support or surface or does not fix with another. In embodiments, loose molecule is the solute (such as nucleic acid) by flowable medium.Illustrative medium In transfer or it is mobile can be it is intracorporal other by diffusion, convection current, turbulent flow, stirring, Brownian movement, advection, electric current or liquid The transfer of any other point of the molecular motion from any first point in continuous phase into fluid communication or identical continuous phase Or it is mobile.For example, the loose nucleic acid of significant quantity is transferred to flowable from a fixed site in flowable medium Medium identical continuous phase in or be in fluid communication with the first fixed site another fix site.Optionally, medium The rate of transfer or the movement of the rate and water amplifying nucleic acid of transfer or the movement of object amplifying nucleic acid is comparable.In some instances, The condition of selection is medium condition experienced during amplification.The condition of selection may or may not allow flowable medium Keep substantially stationary.Condition may or may not make the effective mixing of flowable medium experience, stirring or shake.Medium It is temporarily, at least flowable optionally during amplification.Such as medium is at least one pre- amplification and/or expansion of selection It is flowable under the conditions of increasing.Optionally, flowable medium does not prevent the mixed of different loose nucleic acid not substantively The transfer of conjunction or loose nucleic acid between the different zones of the continuous phase of flowable medium.The movement or transfer of nucleic acid Such as it can be caused by diffusion or convection current.If loose nucleic acid is connecting between different fixation sites or entirely after amplification It cannot propagate or move in continuous phase, then optionally think that medium is not flowable.Generally, it can be flowed during amplification period Loose nucleic acid (such as template or amplicon) is not limited in the effective coverage of reaction capacity substantially by dynamic medium Or it is limited in fixed position.Optionally, can by a variety of methods or the condition by changing flowable medium come It becomes not flowable.It optionally, is flowable if medium is liquid or is not semisolid.Such as The mobility of fruit medium with pure water be it is comparable, then it is believed that it is flowable.In other embodiments, if medium Object is the fluid that there is no polymer, or if the viscosity coefficient of its viscosity coefficient and pure water is similar, it is believed that The medium is flowable.

As it will appreciated by a person of ordinary skill, referring to for template, template polynucleotide, extension probes, primer etc. can refer to It is group or library rather than the individual molecule of substantially the same nucleic acid molecules in relevant part.For example, " template " can refer to it is multiple Substantially the same template molecule；" probe " can refer to multiple substantially the same probe molecules etc..It is one or more positions On be degeneracy probe in the case where, it will be understood that the sequence of the probe molecule comprising particular probe will be different on degeneracy position, Constituting the sequence of the probe molecule of particular probe can only be substantially the same on nondegenerate position.These in the application Term is intended to provide support to group or molecule.When being intended to refer to single nucleic acid molecules (i.e. a molecule), term " template can be used Molecule ", " probe molecule ", " primer molecule " etc. are replaced.It in some instances, will be it is manifestly intended that substantially the same nucleic acid The plural property of molecular group.

" template ", " oligonucleotides ", " probe ", " primer ", " template ", " nucleic acid " etc. are intended to be interchangeable herein Term.These terms refer to polynucleotides, are not necessarily limited to any length or function.Identical nucleic acid can be according to background It is taken as " template ", " probe " or " primer ", and can be converted between these roles at any time." polynucleotides " are also referred to as " nucleic acid " is by the linear polymer of two or more nucleotide of covalent internucleoside linkage connection or its variant or function Property segment.In these naturally occurring example, internucleoside linkage is usually phosphodiester bond.However, other examples are optional Ground includes other internucleoside linkage such as mercaptan phosphate (phosphorothiolate) keys, and may or may not include phosphoric acid Group.Polynucleotides include double-strand and single-stranded DNA and double-strand and single-stranded RNA, DNA:RNA heterozygote, peptide nucleic acid (PNA) heterozygote between PNA and DNA or RNA, and may also include the modification of known type.Polynucleotides are optionally One or more non-nucleotide moieties are connected to by 5 ' or 3 ' ends for example to mark and other small molecules, macromolecular such as egg White, rouge, sugar and solid or semisolid support.Label includes any part that the detection method detection of selection can be used, and because This detect the nucleotide of connection or polynucleotides can similarly by using the detection method of selection.Optionally, label hair Penetrate optically detectable or visible electromagnetic radiation.In some cases, nucleotide or polynucleotides are not connected to mark, and Directly detect the presence of nucleotide or polynucleotides." nucleotide " refers to nucleotide, nucleosides or its analog.Optionally, appoint Selection of land, nucleotide are N- the or C- glucosides of purine or pyrimidine bases.(such as the dezyribonucleoside comprising 2-deoxy-D-ribose Or the ribonucleotide comprising D-ribose).The example of other analogs includes but is not limited to thiophosphate, phosphoramidate, first Base phosphonate ester, chiral-methyl phosphonates, 2-O- methyl ribonucleotides.Refer to that nucleic acid is answered by any one of these terms When not being interpreted to mean that nucleic acid has the function of any specific activity or property.For example, word " template " does not indicate " template " " primer " or " probe " can not be used as by passing through polymerase duplication or template.

It will be understood that in some instances, the nucleic acid reagent such as template, probe, primer etc. for participating in amplification can be larger core The part of acid molecule, the larger nucleic acid molecule also include another part without identical function.Optionally, other portions Divide and does not have any template, probe or primer function.In some instances, substantially it is hybridized to optionally fixed primer (example On such as fixed site) nucleic acid be considered as " template ".It can be generated before or during amplification from other nucleic acid and participate in template Walking any one or more nucleic acid reagents (template, the chain of fixation, fixation or revocable primer etc.).Optionally by right The nucleic acid being initially introduced into template walking medium carries out one or more modifications to generate and (and not need from input nucleic acid It is same) nucleic acid reagent.It can for example make to input nucleic acid experience restrictive digestion, connection, one or more amplification cycles, become Property, mutation etc. to be to generate the nucleic acid for being used as template, primer etc. during amplification or further amplification.For example, can be by the defeated of double-strand Enter nucleic acid denaturation to generate the first single-chain nucleic acid, first single-chain nucleic acid is optionally for generating the second complementary strand.Such as fruiting period It hopes in this way, then it is believed that the first single-chain nucleic acid our purpose " template " in this article.Alternatively, being produced from the first single-chain nucleic acid The second raw complementary strand can be considered as " template " with our purpose in this article.In another example, template source in Input nucleic acid and not necessarily with input nucleic acid it is identical.For example, template may include the other sequence being not present in input nucleic acid Column.In embodiments, template can be using one or more primers with the 5 ' jags not complementary with input nucleic acid The amplicon generated from input nucleic acid.

As herein with respect to used in two or more polynucleotides, term " hybridization " and its variant refer to wherein institute State any one or more nucleic acid sequences (section that each sequence includes continuous nucleotide residue) in polynucleotides at two or Any process of more individual corresponding position experience base pairings (such as in nucleic acid duplex as hybridized).Optionally, " complete " or " entirely " hybridization may be present between the first and second nucleic acid sequences, wherein each of first nucleic acid sequence Nucleotide residue can undergo base pairing interaction with nucleotide corresponding in the antiparallel position in second nucleotide sequence. In some embodiments, hybridization may include two or more not fully complementary over the whole length or non-base pairings Base pairing between nucleic acid sequence.For example, one of nucleic acid sequence is at least when two nucleic acid sequences undergo base pairing 20% but less than 100% residue and other nucleic acid sequences in residue base pairing when, " part " hybridization occur.Some In embodiment, hybridization includes the base pairing between two nucleic acid sequences, one of nucleic acid sequence at least 50% but it is few In 100% residue and residue base pairing corresponding in other nucleic acid sequences.In some embodiments, a nucleic acid sequence At least 70%, 80%, 90% or 95% but match less than 100% residue with residue base corresponding in other nucleic acid sequences It is right.When at least the 85% of the residue of a nucleic acid sequence with corresponding residue base pairing in other nucleic acid sequences, then it is assumed that Two nucleic acid sequences are " substantially hybridizing ".A nucleic acid molecules are (or in which two substantially longer than another wherein Nucleic acid molecules include be substantially complementary and substantially not complementary region) situation in, even if working as one or two nucleic acid molecules Part when can keep non-hybridized, still two nucleic acid molecules can be described as " hybridization "." non-hybridized " describes one of them The nucleic acid sequence of residue base pairing in the residue less than 20% of nucleic acid sequence and other nucleic acid sequences.In some embodiment party In case, base pairing can occur according to a certain conventional pairing pattern, for example, by parallel nucleotide reversely with each other and/ Or the A-T/U and G-C base that the hydrogen of the specific Watson-Crick type between the nucleobase of polynucleotides position bonds together to form It is right；In other embodiments, base pairing can be occurred by any other pattern, and wherein base pairing is according to established It is carried out with predictable rule.

The hybridization of two or more polynucleotides can be whenever the two or more polynucleotides are appropriate miscellaneous When being contacted under the conditions of friendship.Hybridization conditions include any condition suitable for nucleic acid hybridization；The method hybridized It is well known in the present art with the felicity condition for hybridization.The stringency of hybridization can be influenced by multiple parameters, including to miscellaneous The degree of identity and/or complementarity between the polynucleotides (or any target sequence in polynucleotides) of friendship；To be hybridized The melting temperature of polynucleotides and/or target sequence, is called " T_m"；Parameter such as salt, buffer, pH, temperature, multicore glycosides The GC% content and/or time of acid and primer.Normally, hybridization is in lower temperature and/or raised salinity and reduction Organic solvent concentration under be advantageous.High stringency hybridization conditions will usually need the mutual of the higher degree between two target sequences Benefit property to hybridize, even and if hybridization conditions low strict when two polynucleotides wait hybridize shows low-level complementary Also will promote to hybridize.It can be applied in hybridization step or optionally and during successive washing step or hybridization and optional washing step Hybridization conditions.

The example of high stringency hybridization conditions includes any one or more following: the salt (example of about 0.0165 to about 0.0330 Such as NaCl) concentration；Melting temperature (the T of target sequence (or polynucleotides) to be hybridized_m) under about 5 DEG C to about 10 DEG C of temperature； And/or about 50% or higher concentration of forma.Normally, high stringency hybridization conditions allow with high homology for example >=95% Identity or complementarity sequence between combination.In an exemplary implementation scheme of high stringency hybridization conditions, about 42 DEG C are including 25mM KPO₄The salmon for the ultrasonic treatment that (pH 7.4), 5 × SSC, 5 × Denhardt solution, 50 μ g/mL are denaturalized Smart DNA, 50% formamide, the hybridization of 10% dextran sulfate and 1-15ng/mL double-stranded polynucleotide (or double stranded target sequence) are molten Hybridized in liquid, and is washed at about 65 DEG C using the washing solution comprising 0.2 × SSC and 0.1% sodium dodecyl sulfate.

The example of moderate stringency hybridization condition includes any one or more following: the salt (example of about 0.165 to about 0.330 Such as NaCl) concentration；Melting temperature (the T of target sequence to be hybridized_m) under about 20 DEG C to about 29 DEG C of temperature；And/or about 35% Or lower concentration of forma.Normally, such moderate stringency hybridization conditions permit has high or medium homology example Such as the combination between >=80% identity or the sequence of complementarity.In an exemplary embodiment party of moderate stringency hybridization condition In case, including 25mM KPO at about 42 DEG C₄The ultrasound that (pH 7.4), 5 × SSC, 5 × Denhart solution, 50 μ g/mL are denaturalized The salmon sperm DNA of processing, 50% formamide, 10% dextran sulfate and 1-15ng/mL double-stranded polynucleotide (or double stranded target sequence) Hybridization solution in hybridized, and about 50 DEG C using the washing solution comprising 2 × SSC and 0.1% sodium dodecyl sulfate into Row washing.

The example of hybridization conditions low strict includes following any one or more: the salt of about 0.330 to about 0.825 (such as NaCl) concentration；Melting temperature (the T of target sequence to be hybridized_m) under about 40 DEG C to about 48 DEG C of temperature；And/or about 25% or Lower concentration of forma.Normally, such low stringency condition allow the identity having low homology for example >=50% or Combination between complementary sequence.

Some exemplary conditions suitable for hybridization are included in sodium salt such as NaCl, sodium citrate and/or phosphoric acid Polynucleotides to be hybridized are incubated in the solution of sodium.In some embodiments, hybridizing or washing solution can include about 10-75% Formamide and/or about 0.01-0.7% sodium dodecyl sulfate (SDS).In some embodiments, hybridization solution can be Stingent hybridization solution may include 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate Any combination of (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt solution, 0.1%SDS and/or 10% dextran sulfate. In some embodiments, hybridizing or washing solution may include BSA (can be in bovine serum albumin(BSA)).In some embodiments, Can be at about 20-25 DEG C, or about 25-30 DEG C, or about 30-35 DEG C, or about 35-40 DEG C, or about 40-45 DEG C, or about 45-50 DEG C, or Hybridized or washed under about 50-55 DEG C or higher temperature range.

In some embodiments, hybridization or washing can be subjected to about 1-10 minutes, or about 10-20 minutes, or about 20-30 Minute, or about 30-40 minutes, or about 40-50 minutes, or about 50-60 minutes or longer time range.

It in some embodiments, can be in the pH range of about 5-10, or about pH6-9, or about pH6.5-8, or about pH6.5-7 Under hybridized or washed.

In some embodiments, term " monoclonal " and its variant are used to describe the substantive portion of wherein group member Divide (for example, at least about 50%, typically at least 75%, 80%, 85%, 90%, 95% or 99%) total in nucleotide sequence level There is the polynucleotides group of at least 80% identity.Normally, at least about the 90% of group, normally at least about 95%, it is more logical Often at least about 99%, 99.5% or 99.9%) produced by the amplification of specific polynucleotide sequence or Template Dependent duplication Raw, the specific polynucleotide sequence is present among the substantial portions of the member of monoclonal polynucleotides group.Monoclonal group All members need not be identical from one another or complementary.For example, the different piece of polynucleotide template can be amplified or It replicates to generate the member of resulting monoclonal group；Similarly, a certain number of " mistakes " and/or not exclusively extension can be original Occur during the amplification of template, thus generate its individual member can display sequence otherness among themselves monoclonal group. In some embodiments, at least the 50% of the member of monoclonal group (is used for sequence ratio with referring to nucleic acid sequence based on i.e. Compared with the nucleic acid with determining sequence) be at least 80% same.In some embodiments, group at least 60%, extremely Few 70%, at least 80%, at least 90%, at least 95%, at least 99% or more member includes to have with referring to nucleic acid sequence The sequence of the identity (or complementary) of at least 80%, 85%, 90%, 95%, 97% or 99%.In some embodiments, Occur during the nucleic acid amplification reaction that the horizontal mixing of the low or unsubstantiality of non-homogeneous polynucleotides can be described herein, Therefore substantially monoclonal group may include a small number of different polynucleotides (such as less than 30%, less than 20%, less than 10%, Less than 5%, less than 1%, less than 0.5%, the different polynucleotides less than 0.1% or less than 0.001%).Such as institute herein With, phrase " substantially monoclonal " and its variant, when for one or more polynucleotides groups in use, referring to one or more It is a comprising having the polynucleotides group of at least polynucleotides of 80% identity with original single template, it is described original single Template is used as the group that basis generates substantially monoclonal for clonal expansion.

In some embodiments, at least the 80% of the member of amplicon, normally at least the 90% of the member of amplicon, More generally at least 95%, more generally at least 99% will with the shared identity greater than 90% of polynucleotide template, normally Identity greater than 95%, more often over 97% and more often over 99% identity.Alternatively, the member of amplicon Can with primary template have greater than 90% complementarity, normally be greater than 95% complementarity, more often over 97% it is mutual Benefit property, more often over 99% complementarity.In some embodiments, the substantially member of the nucleic acid group of monoclonal can be Hybridize each other under stringent hybridization condition.

In some embodiments, if amplicon includes polyclonal pollutant few enough by template sequence shadow Detectable signal is generated in any method of loud foranalysis of nucleic acids, then amplicon is referred to as " monoclonal " or " substantially Monoclonal ".For example, " monoclonal " group of polynucleotides may include generating the signal that specific sequencing system can be used to detect Any group of (such as sequencing signal, nucleotide are incorporated to signal etc.).Optional, signal then can be analyzed and deposited with correctly measuring It is the sequence and/or base information of any one or more nucleotide in any polynucleotides of group.For detect and/or The example for analyzing the sequencing system appropriate of such signal includes Ion Torrent sequencing system, such as Ion Torrent PGM^TMSequencing system, including 314,316 and 318 systems and Ion Torrent Proton^TMSequencing system, including Proton I, Proton II and Proton III (Life Technologies, Carlsbad, CA).In some embodiments, Dan Ke Grand amplicon allows accurate sequencing of at least five continuous nucleotide residue on Ion Torrent sequencing system.

As used herein, term " clonal expansion " and its variant refer to and are wherein generated by the amplification of polynucleotide template Substantially any process of the polynucleotides group of monoclonal.In some embodiments of clonal expansion, two or more are expanded A polynucleotide template is to generate the polynucleotides groups of at least two substantially monoclonals.

As used herein, term " connector " includes the multicore comprising DNA, RNA, chimeric rna/dna molecule, its analog Thuja acid or oligonucleotides and normally mean to connect or be bound to during operating process purpose target polynucleotide (such as mould Plate) addition or external sequence.The connection of connector to template optionally occurs before or after template amplification.One In a little embodiments, connector may include sequence in conjunction with primer that is substantially the same with the sequence in corresponding primer or being substantially complementary Column.In some embodiments, the first connector comprising the first primer binding site is connected to an end of linear double-stranded template End, and include that the second connector of the second primer binding site is connected to another end.

As used herein, term " binding partners " includes two molecules or part thereof, is had for mutual specific Binding affinity, and normally by prior to and other molecules combination and be bonded to each other.Normally but not necessarily, specific In conjunction with pair a member some or all structures with some or all structures possessed by another member be it is complementary, In two members being capable of the particularly knot by way of the bonding (optional passes through multiple non-covalent attractions) between complementary structure It is combined.

In some embodiments, the molecule as binding partners includes: biotin (and its derivative) companion in connection Companion's avidin part, streptavidin part (and its derivative)；In conjunction with the His- label of nickel, cobalt or copper；Knot Close cysteine, histidine or the histidine section (histidine patch) of Ni-NTA；In conjunction with maltose-binding protein (MBP) Maltose；Agglutinin-carbohydrate binding partners；Calcium-calbindin (CBP)；Acetylcholine and receptor-acetyl gallbladder Alkali；Albumin A and the anti-FLAG antibody of binding partners；GST and binding partners glutathione；Uracil dna glycosylase (UDG) with Ugi (uracil-DNA glycosylase inhibitor) albumen；The antigen or epitope tag of binding antibody or antibody fragment, in particular Antigen such as digoxigenin, fluorescein, dinitrophenol dinitrophenolate or bromodeoxyribouridine and its respective antibody；Mouse immune globulin with Goat anti mouse immunoglobulin；In conjunction with IgG and albumin A；Receptor-receptor agonist or receptor antagonist；Enzyme-enzyme auxiliary The factor；Enzyme-enzyme inhibition factor；With thyroxine-cortisol.Another binding partners of biotin can be the biology from chicken Plain binding protein (Hytonen et al., BMC Structural Biology 7:8).

Avidin part may include the biotin-binding part of avidin and avidin Any derivative, analog and other unnatural forms.The other forms of avidin part include natural sum The avidin and streptavidin and derivative molecule of recombination, such as nonglycosylated avidin, N- acyl group avidin and truncated streptavidin.For example, avidin part includes avidin 9 White deglycosylation form, streptomyces (Streptomyces) (such as avidin streptomycete (Streptomyces Avidinii)) generate bacterium streptavidin, truncated streptavidin, the avidin of recombination and Streptavidin and natural, deglycosylated and recombination avidin and natural, recombination and truncate Streptavidin derivative, such as N- acyl group avidin, such as N- acetyl group, N- phthalyl and N- Succinyl avidin and commercial product ExtrAvidin^TM、Captavidin^TM、Neutravidin^TMWith Neutralite Avidin^TM。

Embodiment

According to the following example it will be further understood that the embodiment of this teaching content, these embodiments should not be solved It is interpreted as limiting the range of this teaching content in any way.

Embodiment 1

It is anti-that nucleic acid amplification is carried out in the single Continuous Liquid Phase of the total reaction volume of~220 μ L in single reaction vessel It answers.

It will about 420x10 with water in 1.5mL pipe (pipe 1)⁶Globule wash 1 time (vortex/rotation), then in buffer 1 time (vortex/rotation) of middle washing.

It recombinates enzyme source and comes from TwistAmp^TMBasic kit (comes from TwistDx, Cambridge, Great Britain).The sediment through being dehydrated in kit include usvX recombinase, usvY recombinase be loaded into albumen, gp32 albumen, Bsu archaeal dna polymerase, dNTP, ATP, phosphocreatine and creatine kinase.In the rehydration buffer of 120 μ L provided by kit Middle rehydration comes from TwistAmp^TMFour sediments (pipe 2) of Basic kit.Recombination enzyme solutions are vortexed and are rotated, with It is ice-cold afterwards.Two heat blocks are prepared, a setting is arranged in about 68-70 DEG C, one at 40 DEG C.

Supernatant is removed from globule sediment (pipe 1), leaves the liquid of about~20 μ L in bottom.

Reverse primer (100 μM of stostes of 2 μ L) is added to globule pipe (pipe 1), be then vortexed and is rotated.Reverse primer sequence Column: 5 '-ATCCCTGCGTGTCTCCGAC-3.

Biotinylated reverse primer (10 μM of stostes of 2 μ L) is added to globule pipe (pipe 1), be then vortexed and is rotated. Biotinylated reverse primer sequences: 5 ' Bio-ATCCCTGCGTGTCTCCGAC-3 '.

The polynucleotides library (various concentration) of 1 μ L is added to globule pipe (pipe 1), vortex/rotation is placed in ice On.Library concentration changes globule ratio according to the DNA of desired 1:50,1:75,1:200.

The recombination enzymatic mixture (pipe 2 is rebuild in 120 μ L rehydration buffers) of rehydration is added to globule pipe (pipe 1) it, is vortexed, rotates；It is placed on ice.

The exemplary screening agent of the present disclosure of 65 μ L is added to globule pipe, be vortexed and is rotated, is placed on ice.

The ice-cold 280mM Mg- acetate of 11 μ L is added to globule pipe (in centre), then the whirlpool under maximum setting It revolves 3 seconds and puts back to 10 seconds on ice, and incubated 20 minutes on heat block in 40 DEG C.

It will be reacted heat inactivation 10 minutes in heat block in 68 DEG C -70 DEG C.

Reaction tube is filled it up with TE buffer, is vortexed and rotates 3 minutes in the case where (~20KG) is arranged in maximum, removed from globule molten Liquid leaves~100 μ L.Wash repeatedly step twice.

It is primary that globule is washed with recycling solution.

Reaction tube is filled it up with washing buffer, is vortexed and rotates 3 minutes in the case where (~20KG) is arranged in maximum, removed from globule Solution leaves~100 μ L.Wash repeatedly step twice.

After last rotation, solution is reduced to 100 μ L (washing solution).

By the way that biotinylated polynucleotides (are come from the para-magnetic beads that streptavidin has been conjugated The MyOne of Dynabeads^TMBead) in conjunction with being enriched with globule.

The globule of enrichment is loaded into Ion Torrent ion-sensitive chip to go forward side by side the quasi- sequencing reaction of rower.Enrichment The signal portion of globule is measured as the group of the substantially monoclonal of the polynucleotides comprising amplification, such as passes through Ion Torrent PGM^TMWhat the observation of the detectable sequencing signal on sequenator from such globule was confirmed.Analysis sequencing signal is to measure The sequence being present in the amplicon of each such globule.

Embodiment 2

By about 240x10⁶Globule (combining forward primer) (comes from Ion in annealing buffer in 2mL pipe Sequencing kit, such as PN 4482006) in washed once.Removal (in addition to~50 μ L) simultaneously abandons supernatant.100 Globule is resuspended in μ L annealing buffer.

There to be 300bp or 400bp Insert Fragment (about 120-240x10⁶Copy) bar code DNA library with it is washed Globule prehybridization.Library is included in an end and is connected to the connector for being hybridized to forward primer and is connected in another end miscellaneous It hands over to the insetion sequence of the connector of reverse primer.Detected template/globule ratio includes 1:1,0.75:1 and 0.5:1.With moving back Fiery buffer adjusts final volume to 200 μ L.By being vortexed and rotating mixing tube.95-100 DEG C incubation pipe 3 minutes, and 37 DEG C incubate 5 minutes.1mL annealing buffer is added, is being higher than 16,000xG vortex and is rotating pipe 3.5 minutes, abandoning supernatant.Add The 10mM potassium acetate for adding 1mL is being higher than 16,000xG vortex and is rotating pipe 3.5 minutes, abandoning supernatant.Repeat potassium acetate washing Once.Globule (pipe 1) is resuspended in 480 μ L potassium acetates.

It recombinates enzyme source and comes from TwistAmp^TMBasic kit (comes from TwistDx, Cambridge, Great Britain).Component list in the sediment through being dehydrated from Basic kit can be found in embodiments above 1.? 15mL is managed in (pipe 2), and rehydration comes from TwistAmp in the rehydration buffer of 2.88mL provided by kit^TM Basic 96 sediments of kit.

100 μM of reverse primers (revocable primer) of 48 μ L are added to the globule (pipe 1) of washed/prehybridization.It will The biotinylated reverse primer of 10 μM of 48 μ L (revocable primer) is added to the globule of washed/prehybridization, and vortex tube (pipe 1).The content (including library, globule and reverse primer) of pipe 1 is added to pipe 2 (sediment comprising rehydration), it will Pipe 2 is vortexed 5 seconds and is placed on ice.144 μ L T4gp32 albumen (15 μ g/ μ L) is added, is vortexed and is returned on ice.Addition The exemplary screening agent of the present disclosure of 1.56mL, vortex tube are simultaneously returned on ice.It is remained above on ice 5 minutes in reaction Later, 264 μ L magnesium acetates of addition, vortex tube 3 times, every time 3 seconds.In 96 orifice plates that 50 μ L sample equal parts are pre-chilled to ice.In heat 96 orifice plates are incubated 25 minutes at 40 DEG C on circulating instrument (temperature maintains 40 DEG C).

In order to terminate reaction, the 100mM EDTA of 150 μ L is added to each hole.By all reactions together and in height It is centrifuged 3.5 minutes in 16,000xG.Abandon supernatant.Add the Tris/1%SDS of 1mL, vortex tube.It is washed in 1mL OneTouch It washs in solution and washs globule twice.Globule is resuspended in 100 μ L.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected in conjunction with to be enriched with The globule of the copy in library.The globule of enrichment is loaded into Ion Torrent PGM ion-sensitive chip.

According to Ion PGM^TMThe specification of manufacturer in Sequencing 400Kit (User Guide PN4474246B) Carry out standard sequencing reaction.The signal portion of the globule for the enrichment being loaded on chip is measured as the multicore glycosides comprising amplification The group of the substantially monoclonal of acid such as passes through Ion Torrent PGM^TMDetectable sequencing on sequenator from these globules What the observation of signal was confirmed.Sequencing signal, which is analyzed, by Torrent Suite Software is present in these globules to measure Amplicon in sequence.

Sequencing data generate 305bp average read length (Fig. 9), the mass measurement of comparison be 1.16G (AQ17) and 1.07G(AQ20)。

Embodiment 3

By about 250x10⁶Globule (combining forward primer) (comes from Ion Sequencing in 1.5mL annealing buffer Kit, such as PN 4482006) in washed once, be vortexed and rotate 6 minutes in 15,000xG.Supernatant is abandoned, is left in pipe About 50 μ L.

There to be 140bp Insert Fragment (about 50x10⁶Copy) library and washed globule prehybridization.Library includes The connector for being hybridized to the connector of forward primer and being connected in another end and being hybridized to reverse primer is connected to an end Insetion sequence.Library (the 62M stoste of 0.81 μ L) and 0.1mL annealing buffer are added to washed globule and by upper Lower imbibition mixing.Globule/template ratios are about 5:1.92-95 DEG C incubation pipe 7 minutes, and 37 DEG C incubate 10 minutes.Addition 1mL annealing buffer is being higher than 15,000xG vortex and is rotating pipe 6 minutes, abandoning supernatant.The 10mM potassium acetate of 1mL is added, It is being higher than 15,000xG vortex and is rotating pipe 6 minutes, is abandoning supernatant.Repeating potassium acetate washed once and be placed in pipe on ice. The liquid of about 60 μ L is retained in pipe (pipe 1).

It recombinates enzyme source and comes from TwistAmp^TMBasic kit (comes from TwistDx, Cambridge, Great Britain).Component list in the sediment through being dehydrated from Basic kit can be found in embodiments above 1.About Rehydration comes from TwistAmp in the rehydration buffer of 240 μ L provided by kit^TM8 precipitatings of Basic kit Object.Sediment and rehydration buffer are vortexed, rotation and ice-cold.

10 μM of biotinylated reverse primers of 100 μM of reverse primers (revocable primer) of 4 μ L and 1 μ L are (non-solid Fixed primer) it is added to the globule (pipe 1) of washed/prehybridization, and be vortexed and rotate and manage (pipe 1).By the content (packet of pipe 1 Containing library, globule and reverse primer) it is added to pipe 2 (sediment comprising rehydration), the vortex of pipe 2 is placed on ice.Addition The exemplary screening agent of the present disclosure of 130 μ L, vortex tube are simultaneously returned on ice.The ice-cold 280mM magnesium acetate of 24 μ L is added, It is vortexed and rotation is managed.Total reaction volume is about 332 μ L.Pipe is incubated 60 minutes at 40 DEG C.

The 100mM EDTA of 1ml is added to terminate reaction.Pipe is vortexed and is rotated 6 minutes in 15,000xG.Abandon supernatant Liquid simultaneously leaves about 20 μ L in pipe.It repeats EDTA and terminates reaction step, vortex and spin step.Add the Tris/1% of 1mL Pipe is vortexed and is rotated 6 minutes in 15,000xG by SDS.It abandons supernatant and leaves about 50 μ L in pipe.In 1mLOneTouch It washs in solution by being vortexed and rotating washing globule, about 100 μ L is left in pipe.All reactions are gathered and 15, 000xG rotates 6 minutes, abandons supernatant, about 100 μ L are left in pipe.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected in conjunction with to be enriched with The globule of the copy in library.The globule of enrichment is loaded into Ion Torrent Proton I^TMIon-sensitive chip.

According to Ion PI^TMThe explanation of manufacturer in Sequencing 200Kit (User Guide PN MAN0007491) Book carries out standard sequencing reaction.The signal portion of the globule for the enrichment being loaded on chip is measured as the multicore comprising amplification The group of the substantially monoclonal of thuja acid such as passes through Ion Torrent Proton^TMIt is detectable from these globules on sequenator The observation of sequencing signal confirmed.Sequencing signal, which is analyzed, by Torrent Suite Software is present in this to measure Sequence in the amplicon of a little globules.

Carry out sequencing operation twice.Sequencing data runs the average read length (Figure 10) for generating 96bp in first time, Second of operation generates the reading length (Figure 11) of 94bp.The mass measurement of comparison first time operation be 1.76G (AQ17) and 1.43G (AQ20) is 1.48G (AQ17) and 1.17G (AQ20) in second of operation.

Embodiment 4:

By globule (combining forward primer) (103x10 of 20 μ L⁶/ μ L) and the 10mM potassium acetate of 440 μ L and the 1M of 3 μ L Tris (pH 8) mixing.Globule is mixed by being vortexed and rotating.

16 μ L are denaturalized by mixing with the NaOH of 2 μ L has 200bp Insert Fragment (about 199x10⁶Copy) text Library is vortexed and rotates, and allows to stand 1 minute.By add 440 μ L 10mM potassium acetate and 3 μ L 1M Tris pH 8 come Neutralization reaction.Library is included in an end and is connected to the connector for being hybridized to forward primer and is connected to hybridization in another end To the insetion sequence of the connector of reverse primer.

Globule is added to the library of denaturation.Globule/template ratios are about 10:1 (200,000,000,000 globules: 200,000,000 libraries).It is vortexed Pipe, and allow be stored at room temperature 5 minutes (pipe 1).

It recombinates enzyme source and comes from TwistAmp^TMBasic kit (comes from TwistDx, Cambridge, Great Britain).Component list in the sediment through being dehydrated from Basic kit can be found in embodiments above 1.? 15mL is managed in (pipe 2), and rehydration comes from TwistAmp in the rehydration buffer of 3mL provided by kit^TMBasic examination 96 sediments (pipe 2) of agent box.

100 μM of biotinylated reverse primers of 100 μM of reverse primers (revocable primer) of 24 μ L and 2 μ L are (non- Fixed primer) it is added to the globule of washed/prehybridization, vortex tube (pipe 1) and ice-cold tube.By the present disclosure of 1.6mL Exemplary screening agent be added to pipe 2, by pipe be vortexed 5 seconds, manually overturn/rotate 10 seconds, be vortexed 5 seconds, be placed on ice.It will 260 μ L 280mM magnesium acetates are added to pipe 2, and pipe is vortexed 5 seconds, manually overturn/rotate (vortex and overturning/rotation 3 in 10 seconds It is secondary), it is placed on ice.In 96 orifice plates that 50 μ L sample equal parts are pre-chilled to ice.96 orifice plates are incubated 60 minutes at 40 DEG C.

The 200mM EDTA for adding 100 μ L is reacted to each hole with terminating.All reactions are gathered and in most high speed Centrifugation 7 minutes.Abandon supernatant.Sediment is resuspended in the 1mL recycling buffer with 1%SDS, is vortexed 30 seconds, and most High speed rotation 6 minutes.After each rotation, pipe is reduced half by the content by merging two pipes.With 1%SDS's Sediment is resuspended in 1mL recycling buffer, is vortexed 30 seconds, and is rotated 7 minutes in 1550rpm.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected in conjunction with to be enriched with The globule of the copy in library.During enriching step, ES- washing buffer is replaced with the recycling buffer with 0.1%SDS. Finally globule is resuspended in 1mL water and is reduced to 100 μ L.The globule of enrichment is loaded into Ion Torrent Proton I^TM Ion-sensitive chip.

Sequencing data generates the average read length (Figure 12) of 144bp, and the mass measurement of comparison is 4G (AQ17).

Embodiment 5

In dH₂By being vortexed and rotating washing 375x10 in O⁶Globule (combining forward primer).Removal supernatant (removes Outside~50 μ L).By DNA library (about 75x10⁶A molecule) it is added to washed globule.By reverse primer (the non-life of about 4 μ L Object element) and the biotinylated reverse primer of 0.4 μ L be added to globule.The fusion forward primer of about 0.8 μ L is added to Globule.The magnesium acetate of 40 μ L is added to globule (final concentration 14mM).By dH₂O is added to the final totality of globule to 320 μ L Product.

It recombinates enzyme source and comes from TwistAmp^TMBasic kit (comes from TwistDx, Cambridge, Great Britain).Component list in the sediment through being dehydrated from Basic kit can be found in embodiments above 1.In list In only pipe, rehydration comes from TwistAmp in the rehydration buffer of 488 μ L provided by kit^TMBasic kit 16 sediments.For the library 400bp, the T4gp32 albumen (final concentration of 0.2mg) of 0.25mg/ml is added, for 600bp The T4gp32 albumen (final concentration of 0.4mg) of 0.5mg/ml is added in library.Vortex tube is to mix and rotate.

The content of bead mixture is added to recombinase pipe, be vortexed and is rotated.By globule/recombination enzymatic mixture and in advance Cold oil is transferred to pipe, and in Ion Torrent OneTouch^TMIt is collected on 10 microns of Sterlitech filters on device.Root Emulsion is generated and broken according to the specification of manufacturer.

By with the paramagnetic streptavidin globule (MyOne from Dynabeads^TMGlobule) it is connected in conjunction with to be enriched with The globule (or omitting enriching step) of the copy in library.The globule of enrichment is loaded into Ion Torrent ion-sensitive core Piece is gone forward side by side the quasi- sequencing reaction of rower.The signal portion of the globule of enrichment is measured as the substantially single of the polynucleotides comprising amplification The group of clone such as passes through Ion Torrent PGM^TMThe observation institute of detectable sequencing signal on sequenator from these globules It confirms.Analysis sequencing signal is to measure the sequence being present in the amplicon of such globule.

Embodiment 6

It will about 120x10 with annealing buffer⁶Globule (combining forward primer) washed once.It removes supernatant and (removes~50 Outside μ L).Washed globule is resuspended in 100 μ L annealing buffers.By the about 60x10 of DNA library⁶Molecule is added to globule. Final volume is adjusted to 200 μ L with annealing buffer.By being vortexed and rotating mixing globule and library.Globule/library is mixed Object is heated to 95-100 DEG C and is kept for 3 minutes, then incubates 5 minutes at 37 DEG C.

The 10mM potassium acetate of 1mL is added to globule/library mixture, be then vortexed and is rotated.Abandon supernatant.It repeats Potassium acetate washed once.Globule/DNA is resuspended in 120 μ L potassium acetates.

It recombinates enzyme source and comes from TwistAmp^TMBasic kit (comes from TwistDx, Cambridge, Great Britain).Component list in the sediment through being dehydrated from Basic kit can be found in embodiments above 1.In list In only pipe, rehydration comes from TwistAmp in the rehydration buffer of 720 μ L provided by kit^TMBasic kit 24 sediments.The dNTP mixture (every kind of dNTP comprising 10mM) of 54 other μ L is added to TwistAmp^TMMixing Object.

In third pipe, by the streptavidin (500 μM) of 3 μ L and the biotinylated reverse primer (100 of 12 μ L μM) mixing, it is subsequently transferred to globule/library pipe.Recombination enzymatic mixture is added to globule/library pipe, is then vortexed to mix And it is ice-cold.27 μ L T4gp32 albumen (15/ μ g/ μ L) are added to globule/library pipe, are vortexed to mix.By the example of 390 μ L Property screening agent be added to globule/library pipe, reverse to manage and be simultaneously vortexed to mix, ice-cold at least 5 minutes.80 μ L magnesium acetates are added to Globule/library pipe, and pipe is vortexed, rotation and at least 10 seconds ice-cold.In 40 DEG C of incubation globule/library pipes 40 minutes.By adding The EDTA (250mM) of 500 μ L is added to terminate reaction.Pipe is rotated 3 minutes in the case where being higher than 18,000xG.Supernatant is abandoned, 1mL's Sediment is resuspended in TE with 1%SDS.Globule is washed twice in 1mLOneTouch washing solution.Globule is resuspended in In 100 μ L.Sediment is resuspended by upper and lower imbibition, and passes through addition 2mL Ion Torrent OneTouch^TMWash solution Washing.It is primary to wash repeatedly step.Sediment is resuspended in 300 μ L melt-off solution (melt-off solution), and is shaking It is lower to incubate 5 minutes.

Embodiment 7

Nucleic acid amplification is carried out on Ion sequence testing chip.Template walking amplified reaction is carried out first on chip, is then carried out The amplified reaction of recombinase-mediated.

Prepare Ion Torrent PGM^TMSequence testing chip is to include low T_MSingle-stranded primer, the primer its 5 ' end connect It is connected to the bottom in hole.Fixed primer includes polyA (30) sequence.

Double-stranded DNA template includes the prominent sequence of the single stranded end with polyT (30) sequence.

With polymer treatment Ion Torrent PGM^TMSequence testing chip is to generate matrix in the bottom in hole.Capture primer connection To matrix.

Ion Torrent PGM is pre-washed with the buffer containing TE^TMSequence testing chip is primary, and is dried in vacuo.

40 microlitres of solution is mixed and is loaded on chip.The final concentration of solution includes: coming from New England 1X isothermal buffer, the 1.6mM MgSO of Biolabs₄, 3mM dNTP, 1U/uL Bst polymerase (come from New England Biolabs), the water of 0.1nM template and nuclease free is to 40uL volume.Chip is centrifuged 5 minutes and is incubated 30 minutes at 37 DEG C. It is dried in vacuo chip.

Template walking amplification: 40 microlitres of template walking solution is loaded on chip.The final concentration of template walking solution Include: 1X isothermal buffer, 3.6mM MgSO from New England Biolabs₄, 5mM dNTP, 2uM it is soluble single-stranded The water of primer, 6U/uL Bst polymerase (come from New England Biolabs) and nuclease free is to 40uL volume.By chip It is centrifuged and is incubated 30 minutes at 60 DEG C.Buffer with 1X containing TE washs chip once and is dried in vacuo.

The amplification of recombinase-mediated: the recombination enzyme source of the present embodiment comes from TwistAmp^TMBasic kit (comes from TwistDx,Cambridge,Great Britain).Component list in the sediment through being dehydrated from Basic kit It can be found in embodiments above 1.50 microlitres of amplification reaction mixtures (including recombinase) are loaded on chip.Amplified reaction Mixture includes: coming from TwistAmp^TMThe one of Basic kit (coming from TwistDx, Cambridge, Great Britain) A sediment comes from TwistAmp^TMThe 30uL rehydration buffer of Basic kit hybridizes with a connector of DNA profiling 2uM soluble primer and nuclease free water to 50uL total volume.Chip is centrifuged 2 minutes.By 2 microlitres of magnesium acetate (280mM stoste) is added to chip.Chip is centrifuged 2 minutes, is then incubated 1 hour at 40 DEG C.

Chip continuously is washed with 0.5M EDTA (pH 8), the buffer containing TE, 1%SDS, and is washed with washing solution 2X。

The basic of the polynucleotides that most hole includes amplification is determined using the comparison map that the color of chip encodes The group of upper monoclonal.

According to Ion PI^TMThe explanation of manufacturer in Sequencing 200Kit (User Guide PN MAN0007491) Book carries out standard sequencing reaction.Sequencing signal, which is analyzed, by Torrent Suite Software is present in these globules to measure Amplicon in sequence.The average read length of sequencing data generation 151bp.

Embodiment 8

Directly in Ion Torrent PGM in the presence of recombinase^TMOn sequence testing chip under isothermal conditions into Row nucleic acid amplification.The hydrogel of polymerization is placed in PGM^TMIn the hole of chip.

The recombination enzyme source of the present embodiment comes from TwistAmp^TMBasic kit (TwistDx, Cambridge are come from, Great Britain).Component list in the sediment through being dehydrated from Basic kit can be found in embodiments above 1。

Using thermal denaturation method or recombination enzyme method (as described above) denatured polynucleotide template, nucleic acid amplification is then carried out Step.

(A) polynucleotide template library thermal denaturation method: is diluted to the final volume of 60 μ L in annealing buffer.Dilution The template merging Ion Torrent Proton for being intended to copy about 5^TMSequence testing chip (about 600-650x10⁶Each of a hole) Kong Zhong.It washs that chip is primary with annealing buffer, is placed on the thermal cycler for being arranged in 40 DEG C.By 1:1 annealing buffer pair The equal portions of 100 μ L of the ratio of water are mixed and are preheated at 95 DEG C.By template library by being placed on chip and incubating 2 at 95 DEG C Minute is denaturalized.Buffer/aqueous mixtures (being preheated to 95 DEG C) are drawn in flow cell.Chip is transferred to 40 DEG C of thermal cycles Instrument simultaneously incubates 5 minutes.Chip is transferred to experimental bench (about 25 DEG C).Chip is washed with the annealing buffer of 100 μ L.Chip is set In on 4 DEG C of thermal cyclers.Following step is optional: being buffered in the water of 20 μ L and the rehydration of 30 μ L provided by kit Rehydration comes from TwistAmp in liquid^TM1 sediment of Basic kit.Violent vortex precipitation mixture is to dissolve precipitating Object.The precipitation mixture of 50 μ L is loaded on chip.It was preloaded at incubation at room temperature chip at least 1 minute with allowing recombinase to combine Enter the primer in the hole of chip.

(B) it recombinase denaturation method: is washed with annealing/aqueous mixtures (ratio of the 1:1 of annealing buffer and water) of 150 μ L Wash Ion Torrent Proton^TMSequence testing chip (about 600-650x10⁶A hole).Chip is placed on 40 DEG C of thermal cyclers.? Rehydration comes from TwistAmp in the rehydration buffer of 60 μ L provided by kit^TM2 sediments of Basic kit. Polynucleotide template library is diluted to the final volume of 50 μ L in annealing buffer.Dilution is intended to the template for copying about 5 It is placed in Ion Torrent Proton^TMIn each hole of sequence testing chip.The diluted template of total volume is added to rehydration Precipitation mixture.Volume is adjusted to 100 μ L with water.Vortex precipitating/template is to mix and rotate.Precipitating/template will be mixed Object is loaded into Ion Torrent Proton^TMSequence testing chip (being placed on 40 DEG C of thermal cyclers) simultaneously incubates 20 minutes.By chip from Thermal cycler is removed and is stood at room temperature.

Following to carry out nucleic acid amplification: all reagents are kept on ice.Delay in the rehydration of 30 μ L provided by kit Rehydration comes from TwistAmp in 100 μM of reversed amplimers of fliud flushing, the nuclease-free water of 16 μ L and 1 μ L^TMBasic reagent 1 sediment of box.By being vortexed and rotating dissolution sediment.Before being loaded into chip immediately, by 280 μM of vinegar of 3 μ L Sour magnesium is added to precipitation mixture.Entire precipitation mixture is loaded on chip and is incubated 1 hour at 40 DEG C.

Amplified reaction is terminated by washing chip with 0.1M EDTA (pH 8).Solution washing chip is washed with chip.With 1%SDS washs chip.Solution washing chip is washed twice with TEX.

Chip is prepared for being sequenced: washing chip using melt-off solution.Chip 3 times are washed with annealing buffer to be placed in On 40 DEG C of thermal cyclers.Sequencing primer is prepared in individual pipe: by 50% annealing buffer and 50% sequencing primer of 80 μ L Mixing, is then preheated to 95 DEG C.It prepares the mixture of the 1:1 of annealing buffer and water and is preheated to 95 DEG C.With preheating Annealing/water buffer wash chip.The preheating primer mixture of 80 μ L is loaded into chip.Divide in 40 DEG C of incubation chips 5 Clock.It is primary with annealing/water buffer washs chip.In individual pipe, by annealing/water of the sequencing polymerase and 57 μ L of 6 μ L Mixture mixing, and be loaded on chip.

Standard sequencing reaction is carried out according to the manufacturer's instructions.

Sequence table

<110> LI, Chieh-Yuan

RUFF, David W.

CHEN, Shiaw-Min

O'NEIL, Jennifer

KASINSKAS, Rachel

ROTHBERG, Jonathan M.

<120>nucleic acid amplification

<130> LT00718 PCT

<140> PCT/US2013/032598

<141> 2013-03-15

<150> 61/635,584

<151> 2012-04-19

<150> 61/699,810

<151> 2012-09-11

<150> 61/767,766

<151> 2013-03-14

<150> 61/792,247

<151> 2013-03-15

<160> 1

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>primer synthesized

<400> 1

atccctgcgt gtctccgac 19

Claims

1. the method for being used for nucleic acid amplification, comprising:

(a) at least two polynucleotides are distributed into the array of reaction chamber, this is by introducing the single polynucleotides At least two reaction chambers carry out；With

(b) nucleic acid of at least two substantially monoclonals is formed by expanding at least two indoor polynucleotides of reaction Group, wherein at least two reaction chamber is in fluid communication with each other during amplification.

2. the method for claim 1 wherein at least two polynucleotides to have different sequences.

3. the method for claim 1 wherein during amplification in the case where being not exclusively denaturalized at least two polynucleotides Carry out the amplification.

4. the method for claim 1 wherein the amplification includes: to close at least two polynucleotides with comprising being used for nucleic acid At reagent single reaction mixture contact.

5. the method for claim 1 wherein the amplification includes: by the recombination at least two polynucleotides and reaction chamber Enzyme contact.

6. the method for claim 1 wherein at least two reaction chambers to be operationally coupled to sensor.

7. method for claim 6, wherein the sensor can detect the presence that reaction chamber inner nucleotide is incorporated to by-product.

8. method for claim 6, wherein the reaction chamber includes conformally to be placed in the indoor hydrophilic polymer matrix of reaction.

9. method for claim 8, wherein the hydrophilic polymer matrix matter includes aquogel polymer matrix.

10. method for claim 8, wherein the hydrophilic polymer matrix matter is the polymer substrate of in-situ solidifying.