WO2024259415A2 - Lamp universelle pour capturer des acides nucléiques non apparentés dans des concatémères lamp - Google Patents

Lamp universelle pour capturer des acides nucléiques non apparentés dans des concatémères lamp Download PDF

Info

Publication number
WO2024259415A2
WO2024259415A2 PCT/US2024/034311 US2024034311W WO2024259415A2 WO 2024259415 A2 WO2024259415 A2 WO 2024259415A2 US 2024034311 W US2024034311 W US 2024034311W WO 2024259415 A2 WO2024259415 A2 WO 2024259415A2
Authority
WO
WIPO (PCT)
Prior art keywords
region
lamp
universal
sequence
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/034311
Other languages
English (en)
Other versions
WO2024259415A3 (fr
Inventor
Andrew Ellington
Sanchita BHADRA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Texas System
University of Texas at Austin
Original Assignee
University of Texas System
University of Texas at Austin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Texas System, University of Texas at Austin filed Critical University of Texas System
Publication of WO2024259415A2 publication Critical patent/WO2024259415A2/fr
Publication of WO2024259415A3 publication Critical patent/WO2024259415A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • LAMP loop-mediated isothermal amplification
  • a target nucleic acid sequence comprising: a) providing a target nucleic acid sequence, wherein said target nucleic acid sequence comprises a target-specific hybridization region; b) carrying out a nucleic acid amplification step using a forward primer and a reverse primer, wherein both primers comprise a universal LAMP handle, wherein said universal LAMP handles comprise a universal amplification support region comprising one or more regions for hybridization of universal priming sequences, thereby producing a universal LAMP target sequence, wherein said universal LAMP target sequence comprises a target sequence flanked by universal handles on both a 3’ and 5’ terminus of the target sequence; and c) carrying out amplification using LAMP with universal priming sequences which recognize the universal support region, thereby producing concatameric amplicons comprising amplified copies of the target nucleic acid sequence.
  • LAMP universal loop-mediated isothermal amplification
  • kits comprising: a) a forward universal LAMP handle comprising a forward nucleic acid extension sequence (F-strand), wherein the F-strand comprises in 3’ to 5’ order, a hybridization region to a target nucleic acid sequence, an F1 region, a loop region, an F2 region, and an F3 region, and further wherein the hybridization region hybridizes with the target sequence, and the F1, F2, and F3 regions are primer-interacting sequences; and b) a reverse universal LAMP handle comprising a reverse nucleic acid extension sequence (B-strand), wherein the B-strand comprises in 3’ to 5’ order, a hybridization region, a B1 region, a loop region, a B2 region, and a B3 region, and further wherein the hybridization region hybridizes with the target sequence and the B1, B2, and B3 regions are primer-interacting sequences.
  • F-strand forward nucleic acid extension sequence
  • B-strand reverse nucleic acid extension sequence
  • the described universal LAMP can be used to capture almost any target nucleic acid sequence (few tens to hundreds of base pairs) within LAMP concatemers without having to design new LAMP primers. It is noted that a universal handle primer with a LAMP handle can be broken into two shorter primers with overlaps, instead of one long primer. In this case, nucleic acid amplification uses all four primers to generate the universal LAMP template.
  • Figure 2 shows PCR to LAMPSeq for Chlamydia trachomatis using SRB sequences as the universal LAMP amplicon.
  • Figure 3 shows BLAST results of PCR to LAMPSeq for Chlamydia trachomatis using SRB sequences as the universal LAMP amplicon.
  • Figure 4 shows alignment reads from nanopore sequencing of the PCR to LAMPSeq for Chlamydia trachomatis using SRB sequence as the universal LAMP amplicon (SEQ ID NOS 1 and 2).
  • Figure 5 shows alignment reads from nanopore sequencing of the PCR to LAMPSeq for Chlamydia trachomatis using SRB sequence as the universal LAMP amplicon (SEQ ID NOS: 1 and 2).
  • F igure 6 shows BLAST query of concatemer hits C. trachomatis.
  • Figure 7 shows PCR to LAMPSeq for Fusobacterium nucleatum FN1868.
  • Figure 8 shows BLAST results of PCR to LAMPSeq for Fusobacterium nucleatum FN1868.
  • Figure 9 shows alignment reads from nanopore sequencing of the PCR to LAMPSeq for Fusobacterium nucleatum FN1868 (SEQ ID NOS: 3 and 4).
  • Figure 10 shows alignment reads from nanopore sequencing of the PCR to LAMPSeq for Fusobacterium nucleatum FN1868 (SEQ ID NOS: 3 and 4).
  • F igure 11 shows BLAST query of concatemer hits F. nucleatum.
  • Figure 12 shows PCR to LAMPSeq for MERS-CoV upE using Ebolavirus LAMP amplicon-based universal handles.
  • Figure 13 shows BLAST results of PCR to LAMPSeq for MERS-CoV upE using Ebolavirus LAMP amplicon-based universal handles.
  • the concatemer units of the captured target(s) may be released and recombined into long concatemers without the intervening universal LAMP.
  • Figure 21 shows long LAMP can generate concatemeric targets for use as novel biomaterials in nicking enzyme use.
  • nicking enzyme sites in F1 or B1 regions of the universal LAMP amplicon the concatemer units of the captured target(s) can be released as single stranded DNA upon denaturation.
  • These ssDNA in turn can fold into hairpins and intermolecular double stranded DNA networks upon cooling. Reamplification via self-priming 3’-end hairpins (or primer addition) is feasible.
  • Figure 22 shows long LAMP can generate concatemeric targets for use as novel biomaterials in concatemer transcription and translation.
  • Long LAMP can be used to capture and concatemerize transcription units designed to yield unit transcripts or very long concatemeric RNA.
  • These RNA can have various uses, such as preparation of RNA hydrogels, probing single molecules, preparation of double stranded RNA, and translation of novel concatemeric proteins. They can also have various shapes due to hybridization between inverted repeats.
  • Figure 23A-D shows alternate methods of using the long LAMP method described herein. For example, shown is the use of hairpin primers for isothermal capture of longer targets.
  • Chimeric target-specific primers whose 5’-ends are extended with universal LAMP or DCA hairpin primers can enable capture of longer targets.
  • outer primers similar to outer primers in LAMP, can be used to displace strands synthesized by the hairpin primers to kickstart self-priming hairpin amplicon formation.
  • A shows chimeric hairpin LAMP primers.
  • B shows chimeric hairpin phosphorothioated DCA primers.
  • C shows non-chimeric target-specific toehold hairpin primers.
  • D shows various conformations.
  • each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed.
  • a “self-assembly pathway” is a series of reactions autonomously executed by nucleic acid sequences in the execution of hybridized, detectable nucleic acid sequences.
  • the self-assembly pathway comprises assembly, or hybridization, of nucleic acid sequences.
  • the self-assembly pathway can also comprise one or more disassembly reactions.
  • nucleic acid refers to natural nucleic acids, artificial nucleic acids, analogs thereof, or combinations thereof.
  • Nucleic acids may also include analogs of DNA or RNA having modifications to either the bases or the backbone.
  • nucleic acid includes the use of peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • nucleic acids also includes chimeric molecules.
  • hairpin refers to a structure formed by intramolecular base pairing in a single-stranded polynucleotide ending in an unpaired loop (the “hairpin loop”).
  • hairpins comprise a hairpin loop protected by stems.
  • a hairpin can comprise a first stem region, a hairpin loop region, and a second stem region. The first and second stem regions can hybridize to each other and together form a duplex region.
  • a stem region of a hairpin nucleic acid is a region that hybridizes to a complementary portion of the same nucleic acid to form the duplex stem of a hairpin.
  • the term “hairpin loop” refers to a single stranded region that loops back on itself and is closed by a single base pair.
  • “Interior loop” and “internal loop,” are used interchangeably and refer to a loop closed by two base pairs. The closing base pairs are separate by single stranded regions of zero or more bases.
  • a “bulge loop” is an interior loop where one of the separated single- stranded regions is zero bases in length and the other is greater than zero bases in length.
  • an “initiator” is a molecule that is able to initiate the hybridization of two other nucleic acid sequences.
  • the initiator is also referred to herein as the third nucleic acid sequence, while it facilitates the hybridization of what is referred to herein as the first and second nucleic acid sequences.
  • “Monomers” as used herein refers to individual nucleic acid sequences. For example, monomers are referred to herein as a first nucleic acid sequence, a second nucleic acid sequence, or a third nucleic acid sequence, etc.
  • Attorney Docket No. 10046-528WO1 8141 ELL By “nucleic acid sequence” is meant a nucleic acid which comprises an individual sequence.
  • first, second, or third nucleic acid sequence when referred to, this is meant that the individual nucleotides of each of the first, second, third, etc., nucleic acid sequence are unique and differ from each other. In other words, the first nucleic acid sequence will differ in nucleotide sequences from the second and third, etc. There can be multiple nucleic acid sequences with the same sequence. For instance, when a “first nucleic acid sequence” is referred to, this can include multiple copies of the same sequence, all of which are referred to as a “first nucleic acid sequence.”
  • target nucleic acid sequence or “target sequence” refers to specific nucleic acid sequences of interest to be amplified and/or detected.
  • target nucleic acid can originate from a variety of sources.
  • target nucleic acids can be naturally occurring DNA or RNA isolated from any source, recombinant molecules, cDNA, or synthetic analogs, as known in the art.
  • the target nucleic acid sequence may comprise one or more single-nucleotide polymorphisms (SNPs), allelic variants, and other mutations such as deletion mutations, insertion mutations, point mutations.
  • the target nucleic acid sequence may comprise a junction sequence of a fusion gene, possibly associated with cancer.
  • the target nucleic acid sequence may originate from a microorganism, including specific clones or strains of microorganisms, possibly involved in inducing diseases in human beings and animals.
  • a microorganism including specific clones or strains of microorganisms, possibly involved in inducing diseases in human beings and animals.
  • at least two different nucleic acid sequences are used in self-assembly pathways, although three, four, five, six or more may be used.
  • each nucleic acid sequence comprises at least one domain that is complementary to at least a portion of one other sequence being used for the self-assembly pathway. Individual nucleic acid sequences are discussed in more detail below.
  • domain refers to a portion of a nucleic acid sequence.
  • An “input domain” of a nucleic acid sequence refers to a domain that is configured to receive a signal which initiates a physical and/or chemical change, such as, a for example, a conformational change, of the nucleic acid sequence.
  • an input domain can be an initiator binding domain, an assembly complement domain, or a disassembly complement domain.
  • An “output domain” of a nucleic acid sequence refers to a domain that is configured to confer a signal.
  • the signal can bind a complementary sequence Attorney Docket No. 10046-528WO1 8141 ELL to an input domain.
  • an output domain is configured to confer a signal to an input domain of another nucleic acid sequence.
  • an output domain can be, for example, an assembly domain, or a disassembly domain. In some embodiments, an output domain can be present in an initiator.
  • nucleate as used herein means to begin a process of, for example, a physical and/or chemical change at a discrete point in a system. The term “nucleation” refers to the beginning of physical and/or chemical changes at discrete points in a system.
  • toehold refers to nucleation site of a domain comprising a nucleic acid sequence designed to initiate hybridization of the domain with a complementary nucleic acid sequence. The secondary structure of a nucleic acid sequence may be such that the toehold is exposed or sequestered.
  • the secondary structure of the toehold is such that the toehold is available to hybridize to a complementary nucleic acid (the toehold is “exposed,” or “accessible”), and in other embodiments, the secondary structure of the toehold is such that the toehold is not available to hybridize to a complementary nucleic acid (the toehold is “sequestered,” or “inaccessible”). If the toehold is sequestered or otherwise unavailable, the toehold can be made available by some event such as, for example, the opening of the hairpin of which it is a part of. When exposed, a toehold is configured such that a complementary nucleic acid sequence can nucleate at the toehold.
  • a “propagation region” as used herein refers to a portion of a domain of a first nucleic acid sequence that is configured to hybridize to a complementary second nucleic acid sequence once the toehold of the domain nucleates at an exposed toehold of the second nucleic acid sequence.
  • the propagation region is configured such that an available secondary nucleic acid sequence does not nucleate at the propagation region; rather, the propagation region hybridizes to the second nucleic acid sequence only after nucleation at the toehold of the same domain.
  • nucleic acid sequences can be “metastable.” That is, in the absence of an initiator they are kinetically disfavored from associating with other nucleic acid sequences comprising complementary regions.
  • polymerization and “assembly” are used interchangeably and refer to the association of two or more nucleic acid sequence, or one or more nucleic acid sequences and an initiator, to form a polymer.
  • the “polymer” may comprise covalent bonds, non-covalent bonds or both.
  • a Attorney Docket No. 10046-528WO1 8141 ELL first, second, and third nucleic acid sequence can hybridize sequentially to form a polymer comprising a three-arm branched junction.
  • disassembly refers to the disassociation of an initiator or at least one nucleic acid sequence.
  • reaction graph refers to a representation of assembly (and, optionally, disassembly) pathways that can be translated into molecular executables.
  • flip and “switch” are used interchangeably and refer to a change from one state (e.g., accessible) to another state (e.g., inaccessible).
  • switch is used interchangeably and refer to a change from one state (e.g., accessible) to another state (e.g., inaccessible).
  • “Kinetically trapped” means that the nucleic acid sequences are inaccessible. In other words, a nucleic acid sequence which is “kinetically trapped” is not available for hybridization. For example, a nucleic acid sequence which has formed a hairpin is considered to be kinetically trapped.
  • an “aptamer” is an oligonucleotide that is able to specifically bind an analyte of interest other than by base pair hybridization.
  • Aptamers typically comprise DNA or RNA or a mixture of DNA and RNA.
  • Aptamers may be naturally occurring or made by synthetic or recombinant means.
  • the aptamers are typically single stranded, but may also be double stranded or triple stranded. They may comprise naturally occurring nucleotides, nucleotides that have been modified in some way, such as by chemical modification, and unnatural bases, for example 2-aminopurine. See, for example, U.S. Pat. No. 5,840,867.
  • the aptamers may be chemically modified, for example, by the addition of a label, such as a fluorophore, or by the addition of a molecule that allows the aptamer to be crosslinked to a molecule to which it is bound.
  • a label such as a fluorophore
  • Aptamers are of the same “type” if they have the same sequence or are capable of specific binding to the same molecule.
  • the length of the aptamer will vary, but is typically less than about 100 nucleotides.
  • oligonucleotides refers to oligomers of natural (RNA or DNA) or modified nucleic acid sequences or linkages, including natural and unnatural deoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptide nucleic acid monomers (PNAs), locked nucleotide acids monomers (LNA), and the like and/or combinations thereof, capable of specifically binding to a single-stranded polynucleotide by way of a regular pattern of sequence-to-sequence interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • PNAs peptide nucleic acid monomers
  • LNA locked nucleotide acids monomers
  • nucleic acid sequences are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few base units, e.g., 8-12, to several tens of base units, e.g., 100-200.
  • Suitable oligonucleotides may be prepared by Attorney Docket No. 10046-528WO1 8141 ELL the phosphoramidite method described by Beaucage and Carruthers (Tetrahedron Lett., 22, 1859-1862, 1981), or by the triester method according to Matteucci, et al. (J. Am. Chem.
  • Oligonucleotides (both DNA and RNA) may also be synthesized enzymatically for instance by transcription or strand displacement amplification. Typically, oligonucleotides are single-stranded, but double-stranded or partially double-stranded oligos may also be used in certain embodiments of the invention.
  • An “oligo pair” is a pair of oligos that specifically bind to one another (i.e., are complementary (e.g., perfectly complementary) to one another).
  • complementary and complementarity refer to oligonucleotides related by base-pairing rules.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • a and T or A and U
  • C and G For example, for the sequence “5′-AGT-3′,” the perfectly complementary sequence is “3′-TCA-5′.”
  • complementarity may be computed using online resources, such as, e.g., the NCBI BLAST website (ncbi.nlm.nih.gov/blast/producttable.shtml) and the Oligonucleotides Properties Calculator on the Northwestern University website (basic.northwestem.edu/biotools/oligocalc.html).
  • Two single-stranded RNA or DNA molecules may be considered substantially complementary when the nucleotides of one strand, optimally aligned and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
  • Two single-stranded oligonucleotides are considered perfectly complementary when the nucleotides of one strand, optimally aligned and with appropriate nucleotide insertions or deletions, pair with 100% of the nucleotides of the other strand.
  • substantial complementarity exists when a first oligonucleotide will hybridize under selective hybridization conditions to a second oligonucleotide.
  • Selective hybridization conditions include, but are not limited to, stringent hybridization conditions.
  • Selective hybridization, or substantially complementary hybridization occurs when at least about 65% of the nucleic acid sequences within a first oligonucleotide over a stretch of at least 14 to 25 sequences pair with a perfectly complementary sequences within a second oligonucleotide, preferably at least about 75%, more preferably at least about 90%.
  • the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. See, M. Kanehisa, Nucleic Acids Attorney Docket No. 10046-528WO1 8141 ELL Res. 12, 203 (1984), incorporated herein by reference.
  • hybridization occurs when at least about 65% of the nucleic acid sequences within a first oligonucleotide over a stretch of at least 8 to 12 nucleotides pair with a perfectly complementary nucleic acid sequence within a second oligonucleotide, preferably at least about 75%, more preferably at least about 90%.
  • Stringent hybridization conditions will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM.
  • Hybridization temperatures can be as low as 5° C., and are preferably lower than about 30° C. However, longer fragments may require higher hybridization temperatures for specific hybridization. Hybridization temperatures are generally at least about 2° C.
  • two perfectly matched nucleotide sequences refers to a nucleic acid duplex wherein the two nucleotide strands match according to the Watson-Crick basepair principle, i.e., A-T and C-G pairs in DNA:DNA duplex and A-U and C-G pairs in DNA:RNA or RNA:RNA duplex, and there is no deletion or addition in each of the two strands.
  • mismatch refers to a nucleic acid duplex wherein at least one of the nucleotide base pairs do not form a match according to the Watson-Crick basepair principle.
  • A-C or U-G “pairs” are lined up, which are not capable of forming a basepair.
  • the mismatch can be in a single set of bases, or in two, three, four, five, or more basepairs of the nucleic acid duplex.
  • “complementary to each other over at least a portion of their sequence” means that at least two or more consecutive nucleotide base pairs are complementary to each other. For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotide base pairs can be complementary to each other over the length of the nucleic acid sequence. .
  • substantially hybridized refers to the conditions under which a stable duplex is formed between two nucleic acid sequences, and can be detected. This is discussed in more detail below.
  • melting temperature (“Tm”) refers to the midpoint of the temperature range over which nucleic acid duplex, i.e., DNA:DNA, DNA:RNA and RNA:RNA, is denatured.
  • stringency of hybridization in determining percentage mismatch is as follows: Attorney Docket No.
  • a “significant reduction in background hybridization” means that non-specific hybridization, or hybridization between unintended nucleic acid sequences, is reduced by at least 80%, more preferably by at least 90%, even more preferably by at least 95%, still more preferably by at least 99%.
  • preferentially binds it is meant that a specific binding event between a first and second molecule occurs at least 20 times or more, preferably 50 times or more, more preferably 100 times or more, and even 1000 times or more often than a nonspecific binding event between the first molecule and a molecule that is not the second molecule.
  • a capture moiety can be designed to preferentially bind to a given target agent at least 20 times or more, preferably 50 times or more, more preferably 100 times or more, and even 1000 times or more often than to other molecules in a biological solution.
  • an immobilized binding partner in certain embodiments, will preferentially bind to a target agent, capture moiety, or capture moiety/target agent complex. While not wishing to be limited by applicants present understanding of the invention, it is believed binding will be recognized as existing when the Ka is at 10 7 l/mole or greater, preferably 10 8 l/mole or greater.
  • the binding affinity of 10 7 l/mole or more may be due to (1) a single monoclonal antibody (e.g., large numbers of one kind of antibody) or (2) a plurality of different monoclonal antibodies (e.g., large numbers of each of several different monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use combinations of (1)-(3).
  • the differential in binding affinity may be accomplished by using several different antibodies as per (1)-(3) above and as such some of the antibodies in a mixture could have less than a four-fold difference.
  • an indication that no binding occurs means that the equilibrium or affinity constant Ka is 10 6 l/mole or less.
  • Antibodies may be designed to maximize binding to the intended antigen by designing peptides to Attorney Docket No. 10046-528WO1 8141 ELL specific epitopes that are more accessible to binding, as can be predicted by one skilled in the art.
  • sample in the present specification and claims is used in its broadest sense and can be, by non-limiting example, any sample that is suspected of containing a target agent(s) to be detected. It is meant to include specimens or cultures (e.g., microbiological cultures), and biological and environmental specimens as well as non- biological specimens.
  • Biological samples may comprise animal-derived materials, including fluid (e.g., blood, saliva, urine, lymph, etc.), solid (e.g., stool) or tissue (e.g., buccal, organ- specific, skin, etc.), as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste.
  • Biological samples may be obtained from, e.g., humans, any domestic or wild animals, plants, bacteria or other microorganisms, etc.
  • Environmental samples can include environmental material such as surface matter, soil, water (e.g., contaminated water), air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.
  • a substance is commonly said to be present in “excess” or “molar excess” relative to another component if that component is present at a higher molar concentration than the other component. Often, when present in excess, the component will be present in at least a 10-fold molar excess and commonly at 100-1,000,000 fold molar excess. Those of skill in the art would appreciate and understand the particular degree or amount of excess preferred for any particular reaction or reaction conditions. Such excess is often empirically determined and/or optimized for a particular reaction or reaction conditions.
  • a promoter, a promoter region or promoter element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked.
  • the promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
  • the promoter region includes Attorney Docket No. 10046-528WO1 8141 ELL sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated.
  • “operatively linked or operationally associated” refers to the functional relationship of nucleic acids with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences.
  • operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • consensus ribosome binding sites see, e.g., Kozak, J. Biol.
  • RNA polymerase refers to an enzyme that synthesizes RNA using a DNA or RNA as the template. It is intended to encompass any RNA polymerase with conservative amino acid substitutions that do not substantially alter its activity.
  • reverse transcriptase refers to an enzyme that synthesizes DNA using a RNA as the template. It is intended to encompass any reverse transcriptase with conservative amino acid substitutions that do not substantially alter its activity.
  • Enzymatically produced refers to the production or secondary or tertiary folding of a nucleic acid by an enzyme rather than by chemical synthesis. Enzymatically produced nucleic acids can be made in vitro or in vivo.
  • ribozyme-containing transcription template scaffolds can be engineered to enable enzymatic co-transcriptional synthesis of RNA circuits that can operate without any post-synthetic separation and re- folding of individual circuit components.
  • primer generally refers to an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed.
  • the sequence of nucleotides added Attorney Docket No. 10046-528WO1 8141 ELL during the extension reaction may be determined by the sequence of the template polynucleotide.
  • primers are extended by a DNA polymerase.
  • Primers are generally of a length compatible with their use in synthesis of primer extension products, and usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges.
  • Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18- 40, 20-30, 21-25 and so on, and any length between the stated ranges.
  • the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.
  • the term “primer- interacting sequences” refers to the region of a target nucleic acid sequence where a primer binds. Primers are usually single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primers can first be treated to separate its strands before being used to prepare primer extension products, or simply referred to as extension products. This denaturation step can be effectuated by heat, but may alternatively be carried out using alkali, followed by neutralization.
  • a “primer” is complementary to a polynucleotide template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded bases linked at its 3′ end complementary to the template in the process of DNA synthesis.
  • extension product generally refers to a product of a reaction in which a nucleotide primer is extended by the covalent addition of nucleotides.
  • the nucleotide incorporation can be guided by a template.
  • the nucleotide incorporation can occur without a template (e.g., “template-independent”).
  • an extension product is an amplification product, such as from PCR amplification, rolling circle amplification (RCA), or isothermal amplification.
  • a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe).
  • a barcode can be part of an analyte, or independent of an analyte.
  • a barcode can be attached to an analyte.
  • a particular barcode can be unique relative to other barcodes.
  • target-specific long LAMP primer sets where greater than100 base pairs of target sequence separate the F1 and B1 sites. Furthermore, the universal long LAMP method enables concatemerization of longer stretches of target DNA.
  • target-specific primers that are designed to incorporate a larger target sequence between their F1 and B1 regions have been used. For example, it has been designed to amplify SARS-CoV-2 spike gene. 5-10 of such primer sets can be incorporated in a multiplex reaction that include some OSD probes to provide an immediate yes/no indication of sample positivity. Amplicons in the positive reactions can then be directly sequenced to obtain genomic information about almost the entire viral Spike gene, for example.
  • immobilized concatemers By labeling the 5’-end of the FIP or BIP LAMP primer, immobilized concatemers can be generated. By using long LAMP, desired concatemerized targets can be immobilized on surfaces. Such immobilized amplicons can be useful for various applications such as sequencing. It can also be used for capturing proteins or other molecules that interact with the chosen target sequence, such as DNA, RNA, or aptamer binding proteins and molecules for detection, purification, or assembling hybrid nanostructures and nanomaterials; single molecule detection, for instance by directing the single stranded loop region to bind the desired nucleic acid sequence and using DNA binding domain-FP fusion proteins, FRET probes, or other DNA readout methods to bind the concatemeric target for imaging.
  • target sequence such as DNA, RNA, or aptamer binding proteins and molecules for detection, purification, or assembling hybrid nanostructures and nanomaterials
  • single molecule detection for instance by directing the single stranded loop region to bind the desired nucleic acid sequence and
  • a target nucleic acid sequence comprising: a) providing a target nucleic acid sequence, wherein said target nucleic acid sequence comprises a target-specific hybridization region; b) carrying out a nucleic acid amplification step using a forward primer and a reverse primer, wherein both primers comprise a universal LAMP handle, wherein said universal LAMP handles Attorney Docket No.
  • LAMP loop-mediated isothermal amplification
  • 10046-528WO1 8141 ELL comprise a universal amplification support region comprising one or more regions for hybridization of universal priming sequences, thereby producing a universal LAMP target sequence, wherein said universal LAMP target sequence comprises a target sequence flanked by universal handles on both a 3’ and 5’ terminus of the target sequence; and c) carrying out amplification using LAMP with universal priming sequences which recognize the universal support region, thereby producing concatameric amplicons comprising amplified copies of the target nucleic acid sequence.
  • a target nucleic acid sequence using a universal loop-mediated isothermal amplification (LAMP) target sequence
  • the method comprising: a) providing a target nucleic acid sequence; b) hybridizing a forward universal handle (F-strand) to the target sequence, wherein the F- strand comprises in 3’ to 5’ order, a hybridization region, an F1 region, a loop region, an F2 region, and an F3 region, and further wherein the hybridization region hybridizes with the target sequence, and the F1, F2, and F3 regions are primer-interacting sequences; c) hybridizing a reverse universal handle (B-strand) to the target sequence, wherein the B- strand comprises in 3’ to 5’ order, a hybridization region, a B1 region, a loop region, a B2 region, and a B3 region, and further wherein the hybridization region hybridizes with the target sequence and the B1, B2, and B3 regions are primer-interacting sequences; d)
  • the described method can be used to form concatemeric amplicons.
  • the terms “concatemeric amplicons” or “concatemer” are used interchangeably and generally refer to a polynucleic acid having substantially similar nucleotide sequences linked alternately in a single-stranded chain. These arrayed sequences may be simple repeats of each other, inverted repeats or combinations thereof.
  • the target sequence is 10-1000 nucleotides in length. In some aspects, the target sequence is 50-1000 nucleotides in length. In some aspects, the target sequence is 100-1000 nucleotides in length. In some aspects, the target sequence is 150- 1000 nucleotides in length. In some aspects, the target sequence is 200-1000 nucleotides in length.
  • the target sequence is 250-1000 nucleotides in length. In some aspects, the target sequence is 300-1000 nucleotides in length. In some aspects, the target sequence is 350-1000 nucleotides in length. In some aspects, the target sequence is 400- 1000 nucleotides in length. In some aspects, the target sequence is 450-1000 nucleotides in Attorney Docket No. 10046-528WO1 8141 ELL length. In some aspects, the target sequence is 500-1000 nucleotides in length. In some aspects, the target sequence is 550-1000 nucleotides in length. In some aspects, the target sequence is 600-1000 nucleotides in length. In some aspects, the target sequence is 650- 1000 nucleotides in length.
  • the target sequence is 700-1000 nucleotides in length. In some aspects, the target sequence is 750-1000 nucleotides in length. In some aspects, the target sequence is 20-350 nucleotides in length. In some aspects, the target sequence is 50-350 nucleotides in length. In some aspects, the target sequence is 75-350 nucleotides in length. In some aspects, the target sequence is 100-350 nucleotides in length. In some aspects, the target sequence is 125-350 nucleotides in length. In some aspects, the target sequence is 150-350 nucleotides in length. In some aspects, the target sequence is 175-350 nucleotides in length.
  • the target sequence is 200-350 nucleotides in length. In some aspects, the target sequence is 225-350 nucleotides in length. In some aspects, the target sequence is 250-350 nucleotides in length. In some aspects, the target sequence is 275-350 nucleotides in length. In some aspects, the target sequence is 300-350 nucleotides in length. In some aspects, the F-strand and the B-strand bind the target sequence at the 3’ and 5’ ends of the target, respectively. In some aspects, amplification is carried out using polymerase chain reaction (PCR) or an isothermal reaction.
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR refers to an enzymatic nucleic acid amplification process that involves multiple cycles of denaturing template nucleic acid, annealing primers, and synthesizing a nucleic acid strand complimentary to the template strand. Each cycle typically involves raising and lowering the reaction temperature to provide the proper thermal environment for each step of the cycle.
  • Denaturing template nucleic acid is usually accomplished using high temperature, while annealing primers requires a lower temperature. Synthesis of the nucleic acid complementary to the template strand can typically occur at a temperature between the temperatures used for denaturing and annealing.
  • PCRs include multiplex polymerase chain reactions and single-plex polymerase chain reactions. Suitable PCR techniques are known to one skilled in the art.
  • Isothermal reactions refer to a reaction that provides for the amplification of a nucleic acid using substantially isothermal conditions.
  • substantially isothermal describes reaction conditions that do not require thermocycling.
  • a substantially isothermal reaction may have temperature changes at the beginning and end of an amplification reaction.
  • substantially isothermal reactions include reactions that employ a “hot start” mechanism, in which the Attorney Docket No. 10046-528WO1 8141 ELL reaction mixture is heated to a temperature necessary to activate a component of the reaction mixture and then optionally cooled to a temperature at which a nucleic acid polymerase catalyzes nucleic acid synthesis.
  • substantially isothermal reactions may employ a temperature to deactivate the amplification reaction, a temperature suitable for storage of the amplification products, a temperature for the release of stored reagents, or combination thereof.
  • Thermocycling equipment can be employed to provide reaction conditions comprising a “hot start,” the reaction temperature, a deactivating temperature, or a storage temperature.
  • isothermal reactions include rolling circle amplification (RCA), multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), which involves specific primers to generate quasi circular DNA molecules, helicase-dependent amplification (HDA), and nicking enzyme amplification reaction (NEAR), which involve the generation of nicks in the DNA molecule that are used to prime replication.
  • one-pot synthesis is used to carry out the LAMP method.
  • One- pot synthesis refers to the amplification of nucleic acid in a single reaction vessel or tube.
  • the one-pot synthesis is carried out without the need for intermediate purification or transfer steps.
  • One-pot synthesis approaches can provide a variety of advantages, including, for example, reduced contamination, time and cost efficiency, improved yield, and simplicity.
  • both the step of carrying out nucleic acid amplification, thereby producing a target sequence with universal LAMP handles on both the 3’ and 5’ terminus, thereby producing a universal LAMP target sequence; and the step of carrying out LAMP, thereby producing concatameric amplicons can be carried out in “one pot.”
  • These steps can be carried out at a temperature from 50°C to 90°C; from 55°C to 90°C; from 60°C to 90°C; from 50°C to 85°C; from 50°C to 80°C; from 50°C to 75°C; or at about 65°C.
  • both steps are carried out at the same temperature.
  • the steps are carried out at different temperatures.
  • the hybridization region of the F-strand is at a 3’ terminus of the F- strand. In some aspects, the hybridization region of the B-strand is at a 3’ terminus of the B- strand. In some aspects, both the loop region of both the F-strand and the B-strand comprises a self- complementary hairpin structure which is generated during LAMP. In some aspects, the LAMP reaction step further comprises the steps of: i) providing a forward inner primer (FIP) which complements the F2 region of the target sequence at a 5’ terminus of the FIP, and further wherein said FIP comprises a sequence which complements the F1 region of the universal LAMP target sequence at a 5’ end of the Attorney Docket No.
  • FIP forward inner primer
  • loop primers are used during LAMP amplification. “Loop primers” can be added in conjunction with the other primers used in LAMP to produce significantly faster assays.
  • LAMP uses a total of six primers: two loop-generating primers, two displacement primers and two loop primers. Loop primers are typically positioned between the B2 and B1 sites and the F2 and F1 sites, respectively, and orientated in a particular direction. Some suitable examples of loop primers can be found in WO2000/028082A1, which is hereby incorporated by reference.
  • the target sequence is not circularized prior to amplification.
  • two or more target sequences or sequence variants are detected concurrently, and wherein two or more sets of universal handle sequences are used.
  • the two or more target sequences or sequence variants are present in the same contiguous nucleotide sequence or on two different nucleotide sequences.
  • the two or more target sequences or sequence variants are derived from one organism or from two or more different organisms.
  • target sequences are synthetic or artificial.
  • after amplification at least one extension product is detected.
  • one or more extension products are detected in real time. Attorney Docket No. 10046-528WO1 8141 ELL
  • the product is detected using a hybridization probe.
  • hybridization probes examples include TaqMan hydrolysis probes (U.S. Pat. Nos. 5,210,015, 5,538,848, 5,487,972, and U.S. Pat. No. 5,804,375 (incorporated herein by reference in their entirety)), molecular beacons (U.S. Pat. No. 5,118,801 (incorporated herein by reference in its entirety)), and FRET hybridization probes (WO1997/046707, WO1997/046712, and WO1997/046714 (incorporated herein by reference in their entirety)).
  • the product is detected using fluorescence.
  • Suitable methods for detecting the product using fluorescence include the use of fluorescent labels, such as mutually quenching fluorescent labels, fluorescent label linking agents, enzymes, quenching agents, nucleic acid binding dyes, etc.
  • a signal primer is used to detect amplification.
  • the product is detected by binding of or alterations in the state of nanomaterials or nanoparticles or formation of hydrogels or origami structures via endogenous or exogenous staples or connectors.
  • the product is detected by sequencing.
  • the product is used as template for amplification or transcription and the resulting amplicons or subsequent translation products are detected.
  • kits comprising: a) a forward universal LAMP handle comprising a forward nucleic acid extension sequence (F-strand), wherein the F-strand comprises in 3’ to 5’ order, a hybridization region to a target nucleic acid sequence, an F1 region, a loop region, an F2 region, and an F3 region, and further wherein the hybridization region hybridizes with the target sequence, and the F1, F2, and F3 regions are primer-interacting sequences; and b) a reverse universal LAMP handle comprising a reverse nucleic acid extension sequence (B-strand), wherein the B-strand comprises in 3’ to 5’ order, a hybridization region, a B1 region, a loop region, a B2 region, and a B3 region, and further wherein the hybridization region hybridizes with the target sequence and the B1, B2, and B3 regions are primer-interacting sequences.
  • F-strand forward nucleic acid extension sequence
  • B-strand reverse nucleic acid extension sequence
  • the kit further comprises primers needed for LAMP reaction.
  • the kit further comprises loop primers.
  • loop primers can be added in conjunction with the other primers used in LAMP to produce significantly faster assays. Loop primers are typically positioned between the B2 and B1 sites and the F2 and F1 sites, respectively, and orientated in a particular direction.
  • Non- Attorney Docket No. 10046-528WO1 8141 ELL limiting examples of loop primers suitable for the present kit can be found in WO2000/028082A1, which is hereby incorporated by reference.
  • the kit further comprises reagents for LAMP reaction.
  • LAMP reagents include LAMP primers for one or more target nucleic acids, DNA polymerase with high strand displacement activity (e.g., a DNA polymerase long fragment (LF) of a thermophilic bacterium, such as Bacillus stearothermophilus (Bst), Bacillus Smithii (Bsm), Geobacillus sp.
  • LF DNA polymerase long fragment
  • thermophilic bacterium such as Bacillus stearothermophilus (Bst), Bacillus Smithii (Bsm), Geobacillus sp.
  • dNTPs deoxyribonucleotide triphosphates
  • dATP deoxyadenosine triphosphate
  • dGTP deoxyguanosine triphosphate
  • dCTP deoxycytidine triphosphate
  • dTTP deoxythymidine triphosphate
  • the kit may comprise a plurality of primers for amplification and/or for sequencing nucleic acids isolated from a collected specimen.
  • the kit may provide at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, or more primers.
  • the kit may provide between about 1-3, 1-10, 5-20, 1-1000, 10-500, 20-200, or 50-100 primers.
  • the primers may have 5, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200 or more nucleotides.
  • the primers may have between about 1-8, 5-10, 6-20, 15-30, 20-50, 30-60, 40-80, 50-100, or 10-200 nucleotides.
  • kits refers to any delivery system for delivering materials or reagents for carrying out the method described above.
  • delivery systems can include systems and/or compounds (such as dilutants, surfactants, carriers, or the like) that allow for the storage, transport, or delivery of reaction reagents (e.g., fluorescent labels, such as mutually quenching fluorescent labels, fluorescent label linking agents, enzymes, quenching agents, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • reaction reagents e.g., fluorescent labels, such as mutually quenching fluorescent labels, fluorescent label linking agents, enzymes, quenching agents, etc. in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay etc.
  • kits can include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
  • a first container may contain an enzyme for use in an assay
  • a second or more containers contain mutually quenching fluorescent labels and/or quenching agents.
  • Attorney Docket No. 10046-528WO1 8141 ELL The kit may be provided to users, for example clinical pathology laboratories, a healthcare personnel, a physician, a nurse, a medical care assistance, or a home healthcare assistance.
  • the kit may be intended as a stand-alone solution.
  • the kit may be combined with other kits and instruments.
  • the sample collection device may be a glass slide coated with a functionalized surface.
  • Analyte-specific reagents may be nucleic acid primers and/or probes to detect the panel of target and control nucleic acids.
  • the kit may contain instructions to perform a test using reagents from other vendors. For example, the kit may instruct users to use a Qiagen purification kit to isolate mRNA from a samples collected using a provided sample collection device.
  • the kit may comprise spin column technology (e.g. RNeasy Plus Micro Kit) or magnetic bead-based technology (e.g. ARCTURUS® PicoPure® RNA Isolation Kit, Dynabeads® mRNA DIRECTTM Micro Kit) that may isolate mRNA, total RNA, or total nucleic acids.
  • the disclosed kit may contain a squeegee or cell scraper to enhance sample removal from the provided sample collection device when using a kit or reagents from another vendor.
  • the kit may comprise a storage device for a collected specimen.
  • the storage device may be a sample collection tube, an Eppendorf, a container, or any device that is suitable for storing substances.
  • the kit may contain instructions to use a cDNA synthesis kit from another vendor.
  • the cDNA synthesis kit may contain the SuperScript® III reverse transcriptase, AffinityScript RT, M-MuLV RNase H+ reverse transcriptase, RE3 Reverse Transcriptase, or Quantiscript Reverse Transcriptase with dNTPs in a compatible buffer.
  • the disclosed kit may contain primers to perform cDNA synthesis.
  • the primers disclosed herein may contain a reporter label comprising a tag, fluorescence label, a magnetic bead, or a barcode.
  • the reporter label may be targeted to specific nucleic acids.
  • the reporter label may be used to identify nucleic acids.
  • the primers may be used for sequencing of targeted nucleic acids with or without amplification of the nucleic acids.
  • the sequencing may be any sequencing technologies known in the art.
  • the disclosed kit may provide instructions for performing sequencing using reagents enclosed therein.
  • the disclosed kit may contain instructions to perform cDNA synthesis using random oligonucleotide primers, poly-A primers, or analyte-specific primers.
  • the disclosed kit may contain instructions for the user to amplify RNA or cDNA using enclosed reagents, or reagents provided by another vendor.
  • the instructions may direct users to Attorney Docket No. 10046-528WO1 8141 ELL use enclosed primers to perform analyte-specific amplification using reagents provided by another vendor.
  • the amplification could be performed using PCR, quantitative PCR (QPCR), real-time PCR, digital PCR (dPCR), digital droplet PCR (ddPCR), or isothermal amplification.
  • the real-time PCR reagents from another vendor could consist of Thermo Scientific TaqPathTM qPCR Master Mixes, which can be provided as general purpose reagents.
  • Synthesis of mRNA to cDNA and subsequent amplification can be performed using a kit, for example the TaqPathTM 1-Step RT-qPCR Master Mix. RNA may also be directly used as targets. Analyte-specific probes and fluorescent reporters can also be used. Alternatively, primers without analyte-specific probes can be used, which would be compatible for an intercalating fluorescent reporter, for example a SYBR dye.
  • the products of the amplification methods described herein can be analyzed using a next-generation sequencing platform.
  • the next-generation sequencing platform can be a commercially available platform.
  • platforms include, e.g., platforms for sequencing-by synthesis, ion semiconductor sequencing, pyrosequencing, reversible dye terminator sequencing, sequencing by ligation, single-molecule sequencing, sequencing by hybridization, and nanopore sequencing.
  • Platforms for sequencing by synthesis are available from, e.g., Illumina, 454 Life Sciences, Helicos Biosciences, and Qiagen.
  • Illumina platforms can include, e.g., Illumina's Solexa platform, Illumina's Genome analyzer, which are described in Gudmundsson et al., Genome-wide association and replication studies identity four variants associated with prostate cancer susceptibility. Nat. Genet. 2009 41:1122-1126, Out et al.
  • Platforms for ion semiconductor sequencing include, e.g., the Ion Torrent Personal Genome Machine (PGM) and are described in U.S. Pat. No. 7,948,015, which patent is hereby incorporated in its entirety.
  • Platforms for pyrosequencing include the GS Flex 454 system and are described in U.S. Pat. Nos. 7,211,390; 7,244,559; 7,264,929, which patents are hereby incorporated in their entireties.
  • Platforms and methods for sequencing by ligation include, e.g., the SOLiD sequencing platform and are described in U.S. Pat. No. 5,750,341. Platforms for single- Attorney Docket No.
  • 10046-528WO1 8141 ELL molecule sequencing include the SMRT system from Pacific Bioscience and the Helicos True Single Molecule Sequencing platform.
  • the products produced using the methods described herein can be used as biomatter for various uses known to those of skill in the art. Examples include, but are not limited to, assembly of macro and micromaterials, DNA synthesis, transcript generation, protein generation, information storage, information capture, and information transduction.
  • the methods described herein can be used with barcodes. Barcodes can have a variety of different formats. For example, barcodes can include non-random, semi-random, and/or random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences.
  • One or more barcodes can be used as part of the target sequence, or can be introduced to the LAMP product via primers.
  • a barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner.
  • a barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for identification and/or quantification of individual sequencing-reads (e.g., a barcode can be or can include a unique molecular identifier or “UMI”).
  • Barcodes can spatially-resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”).
  • a barcode includes both a UMI and a spatial barcode.
  • a barcode includes two or more sub-barcodes that together function as a single barcode (e.g., a polynucleotide barcode).
  • a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that may be separated by one or more non-barcode sequences.
  • Detection systems are known in the art, and include optical assays (including fluorescence and chemiluminescent assays), enzymatic assays, radiolabeling, surface plasmon resonance, magnetoresistance, cantilever deflection, surface plasmon resonance, etc.
  • the products made by the methods described herein can be used in additional assay technologies.
  • the surface can be a chip containing synthetic oligonucleotides.
  • the concatemerized nucleic acid (the product of the methods disclosed herein) can be cleaved by digestion. Such means are known to those of skill in the art. This released nucleic acid can then be used as probes for hybridization, or for gene assembly.
  • the concatemers can be barcoded, such that each concatemer comprises a unique sequence.
  • Attorney Docket No. 10046-528WO1 8141 ELL This barcoded concatemerized nucleic acid can be cleaved by digestion and used for gene assembly, as described herein.
  • the products of the method described herein can be attached to solid supports for detection.
  • bead arrays as described below may be used.
  • the present invention provides arrays, each array location comprising at a minimum a covalently attached strand displacement reporter, also referred to herein as a “capture probe”.
  • array herein is meant a plurality of nucleic acid probes in an array format; the size of the array will depend on the composition and end use of the array. Generally, the array will comprise from two to as many as 100,000 or more reporters, depending on the size of the electrodes, as well as the end use of the array. Preferred ranges are from about 2 to about 10,000, with from about 5 to about 1000 being preferred, and from about 10 to about 100 being particularly preferred.
  • the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single capture probe may be made as well.
  • multiple substrates may be used, either of different or identical compositions.
  • large arrays may comprise a plurality of smaller substrates.
  • Nucleic acids arrays are known in the art, and can be classified in a number of ways; both ordered arrays (e.g. the ability to resolve chemistries at discrete sites), and random arrays (e.g. bead arrays) are included.
  • Ordered arrays include, but are not limited to, those made using photolithography techniques (Affymetrix GeneChipTM), spotting techniques (Synteni and others), printing techniques (Hewlett Packard and Rosetta), origami pads, paperfluidics, electrode arrays, three dimensional “gel pad” arrays, etc. Liquid arrays may also be used.
  • substrate or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of nucleic acids.
  • the substrate can comprise a wide variety of materials, as will be appreciated by those in the art. including, but not limited to glass, plastics, polymers, metals, metalloids, ceramics, organics, etc.
  • solid support is a bead, a wide variety of substrates are possible, including magnetic materials, glass, silicon, dextrans, plastics, etc. Chemically derivatized particles, plates, cartridges, tubes, magnetic particles, or other solid phase matrix with specificity to the assay components can also be used.
  • the binding surfaces of microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins Attorney Docket No. 10046-528WO1 8141 ELL such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
  • Platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep- well plates, microfuge tubes, cryovials, square well plates, fitters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes can be accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • the instrumentation can include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, electrochemical and/or electrical impedance analyzers, ultraviolet and visible spectrophotometry detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two- photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • FRET fluorescence resonance energy transfer
  • CCD cameras to capture and transform data and images into quantifiable formats
  • These instruments can fit in a sterile laminar flow or fume hood, or are enclosed, self-contained systems, for cell culture growth and transformation in multi-well plates or tubes and for hazardous operations.
  • the living cells may be grown under controlled growth conditions, with controls for temperature, humidity, and gas for time series of the live cell assays. Automated transformation of cells and automated colony pickers may facilitate rapid screening of desired cells. Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms.
  • the flexible hardware and software allow instrument adaptability for multiple applications.
  • the software program modules allow creation, modification, and running of methods.
  • the system diagnostic modules allow instrument alignment, correct connections, and motor operations.
  • the customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed.
  • the database allows method and parameter storage. Robotic and computer interfaces allow communication between instruments.
  • a biological sample can be a tissue sample, a water sample, an air sample, a food sample or a crop sample.
  • the biological sample analysis detects any one or more of water-born pathogen, air-born pathogen, food-born pathogen or crop-born pathogen.
  • the pathogen detectable by the system and method of the present invention can come from a variety of hosts.
  • the host whether biological or non-biological, should be capable of supporting replication of an infectious agent by allowing the infectious agent to replicate in or on the host. Examples of such hosts include liquid or solid in vitro culture media, cells or tissues of animals, plants or unicellular organisms, whole organisms including mammals such as humans.
  • the kits and methods of the present disclosure can be employed in one of more of the following areas.
  • kits and method of the present invention can be employed in the area of defense against biological weapons.
  • the kits and methods of the present disclosure can be used for point-of-incidence and real-time pathogen-detection.
  • the kits and methods of the present disclosure can be employed in the area of life sciences.
  • the disclosed kits and methods can be used as and with a portable analytical instrument.
  • the kits and method of the present disclosure can be employed in the area of clinical diagnostics.
  • the present disclosure can be used to diagnose and/or identify pathogens by doctors, nurses or untrained users in hospitals, homes or in the field.
  • the present disclosure can be used to genetically identify an individual.
  • genetic disorders and disorders having a genetic component can be diagnosed by employing the system and method of the present disclosure.
  • numerous oncogenes have been identified, including p53, implicated in the development of breast, colorectal and other cancers; c-erbB2, associated with breast cancer development and metastasis; and BRCA1 , involved in 50% of all inherited breast cancers, and also associated with increased risk for prostate and other cancers. Screening for the these genetic markers can be accomplished using the system and methods described herein.
  • the disclosures described herein can be configured or utilized in products or devices that include but are not limited to handheld devices, computer tablets, notebooks, smart phones, implantable devices (implantables), ingestible devices (ingestibles), wearable devices (wearables) and injectable devices (injectables).
  • the device or system can include or be operably coupled to system instructions, e.g., embodied in a computer or computer readable medium.
  • the instructions can control any aspect of the device or system, e.g., to correlate one or more measurements of signal.
  • a system can include a computer operably coupled to the other device components, e.g., through appropriate wiring, or through wireless connections.
  • the computer can include, e.g., instructions that control amplification, e.g., using feedback control as noted above, and/or that specify when images are taken or viewed by the optical train.
  • the computer can receive or convert image information into digital information and/or signal intensity curves as a function of time, determine concentration of a target nucleic acid analyzed by the device, and/or the like.
  • the computer can include instructions for normalizing signal intensity to account for background, e.g., for detecting local background for one or more regions of the array, and for normalizing array signal intensity measurements by correcting for said background.
  • the computer can include instructions for normalizing signal intensity by correcting for variability in array capture nucleic acid spotting, uneven field of view of different regions of the array, or the like.
  • the computer can also comprise a display unit for displaying information received from the signal output unit.
  • EXAMPLES To further illustrate the principles of the present disclosure, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and Attorney Docket No. 10046-528WO1 8141 ELL description of how the compositions, articles, and methods claimed herein are made and evaluated. They are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.); however, some errors and deviations should be accounted for. Unless indicated otherwise, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric.
  • Example 1 Universal Long LAMP Universal LAMP can be used to capture almost any target nucleic acid sequence (few tens to hundreds of base pairs) within LAMP concatemers without having to design new LAMP primers for each target. Referring to Figure 1, target capture is achieved via two chimeric single stranded oligonucleotide primers whose 3’-end portions (shown in pink) are complementary to either the antisense strand (forward target primer) or the sense strand (reverse target primer) of the desired non-cognate target nucleic acid.
  • Binding sites for these two primers on the target nucleic acid may be separated by a few tens to hundreds of base pairs.
  • the longest target amplified efficiently thus far via long LAMP is 231 bp.
  • the 5’-ends of these forward and reverse target primers are extended by addition of the left and right halves, respectively, of a universal LAMP template (structurally organized as 5’-F3-F2-F1-B1-B2-B3-3’ where each F or B domain is a primer interacting sequence).
  • the left half includes the 5’-F3-F2-F1-3’ sequence of the universal template sense strand while the right half is comprised of the 5’-B3c-B2c-B1c-3’ sequence of the antisense strand.
  • LAMP Long LAMP can be operated in a two-pot or a one-pot reaction.
  • the first step is comprised of PCR wherein the two long chimeric primers are used to PCR amplify the target sequence into amplicons of discrete unit size comprising the target sequence flanked by the left and right halves of the LAMP template.
  • these PCR amplicons are used as templates for universal LAMP primers to produce Attorney Docket No. 10046-528WO1 8141 ELL concatemeric LAMP amplicons.
  • the target sequence is isothermally incubated in a single reaction comprising both the chimeric target specific primers and the universal LAMP primers yielding concatemeric end products containing inverted repeats of the target sequence flanked by regions of the universal LAMP amplicon.
  • a study was devised to observe whether universal LAMP amplicons could tolerate long insertions between their F1 and B1 regions. In the experiment, templates flanked by the left and right halves of a universal LAMP template were generated by PCR.
  • Long LAMP was performed in a two-step process where PCR was used in the first step to attached the universal LAMP template left and right halves to the ORF2 amplicon.
  • this PCR product was amplified by LAMP using SRB LAMP primers (FIP+BIP+F3+B3) to create concatemeric LAMP amplicons.
  • SRB LAMP primers FTP+BIP+F3+B3
  • These amplicons were purified using Ampure XP magnetic beads and then prepared for nanopore sequencing using the Oxford nanopore rapid barcoding kit.
  • This kit uses a transposase to simultaneously cleave DNA and attach barcoded sequencing tags to the cleaved ends.
  • the prepared libraries were sequencing using R9 flow cells and the Mk1c platform.
  • FIGS 12-14 show similar two-step long LAMP experimental pipeline as shown in Figures 7-10 with the following differences:
  • the target nucleic acid being tested is a synthetic sequence derived from MERS CoV.
  • the universal LAMP template left and right halves being used here were derived from Ebolavirus LAMP assay instead of the SRB assay used in the previous experiment.
  • Figure 15 shows two additional in-house designed LAMP amplicons, NRP2 and WSP, were tested successfully as universal templates using the two-step experimental process and targets described in the previous slides. Taken together, these data suggest that several different LAMP amplicons have the ability to serve as universal templates. This ability is not restricted to one or two specific amplicons.
  • Figure 16 describes the development of one-step isothermal execution of Long LAMP in one-pot reactions.
  • the NRP2 amplicon was used as the universal template by appending its left and right halves to chimeric target-specific forward and reverse primers (named *.PU.fwd and *.PU.rev).
  • One-pot reactions that included both the target specific chimeric primers and the NRP2 LAMP primer mix comprised of FIP, BIP, F3, and B3 were seeded with either no templates or with 3 ng of a plasmid template.
  • the impact of 40 mM guanidinium hydrochloride, gp32 single stranded DNA binding protein, and additional target-specific primers on the invasion of the chimeric primers was evaluated.
  • coli SNP Figures 2-5) and 18S v9.
  • Corollary non-universal applications include, but are not limited to: single or multiplex LAMP using target-specific LAMP primers designed with long (>100 bp) gaps between the F1 and B1 regions; target-specific OSDs provide an immediate yes/no diagnostic readout; and samples showing a positive result are directly processed for nanopore sequencing.
  • Attorney Docket No. 10046-528WO1 8141 ELL Figure 18 summarizes data from experiments designed to evaluate detection limit of long LAMP.
  • NRP2-based universal long LAMP one-pot isothermal reactions were seeded with either no templates or received indicated amounts of the target plasmid template. Following 2h isothermal amplification at 65 °C, the amplicons were purified and sequenced.
  • the one pot long LAMP reaction can first be subjected to four cycles of PCR using Taq or Vent to allow formation of the initial universal LAMP dumbbell amplicon with the captured target. Subsequent addition of Bst DNA pol and isothermal incubation can yield concatemeric amplicons. Inclusion of denaturants like formamide may promote primer invasion.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Une LAMP universelle peut être utilisée pour capturer des séquences d'acides nucléiques cibles dans des concatémères LAMP sans avoir à concevoir de nouvelles amorces LAMP pour chaque cible. Ces amorces LAMP universelles peuvent être utilisées dans diverses techniques d'amplification et peuvent être incluses dans un kit.
PCT/US2024/034311 2023-06-16 2024-06-17 Lamp universelle pour capturer des acides nucléiques non apparentés dans des concatémères lamp Ceased WO2024259415A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363508633P 2023-06-16 2023-06-16
US63/508,633 2023-06-16

Publications (2)

Publication Number Publication Date
WO2024259415A2 true WO2024259415A2 (fr) 2024-12-19
WO2024259415A3 WO2024259415A3 (fr) 2025-04-03

Family

ID=93852690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/034311 Ceased WO2024259415A2 (fr) 2023-06-16 2024-06-17 Lamp universelle pour capturer des acides nucléiques non apparentés dans des concatémères lamp

Country Status (1)

Country Link
WO (1) WO2024259415A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104334745B (zh) * 2012-05-14 2018-12-04 P·瓦尔霍 用于核酸检测的pcr和环介导的等温扩增的组合技术
US10913973B2 (en) * 2014-09-17 2021-02-09 Board Of Regents, The University Texas System Methods and devices related to toehold-based strand displacement with loop-mediated isothermal amplification

Also Published As

Publication number Publication date
WO2024259415A3 (fr) 2025-04-03

Similar Documents

Publication Publication Date Title
US20200392579A1 (en) Compositions Containing Identifier Sequences on Solid Supports for Nucleic Acid Sequence Analysis
CN1189573C (zh) 多核苷酸序列变异的微阵列型分析
US8673567B2 (en) Method and kit for nucleic acid sequence detection
CN114085896A (zh) 使用非干扰性噪音消除性多核苷酸鉴定标签的多重焦磷酸测序
JP2009505651A (ja) 微生物および抗生物質耐性マーカーの検出の方法およびそのための核酸オリゴヌクレオチド
JP5663491B2 (ja) 標的核酸の検出方法
CN107090480A (zh) 核酸分子的生成
US10538805B2 (en) Quantitative multiplexed identification of nucleic acid targets
US20210363517A1 (en) High throughput amplification and detection of short rna fragments
Uda et al. Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray
JP4628369B2 (ja) 高感度核酸多重解析法
WO2024259415A2 (fr) Lamp universelle pour capturer des acides nucléiques non apparentés dans des concatémères lamp
EP2453022A1 (fr) Procédé de détection ou d'analyse d'une séquence cible dans l'adn génomique
EP4130275A1 (fr) Ensemble d'amorces et sonde pour détection de staphylococcus argenteus
JP5310997B2 (ja) 細菌による汚染の汚染源特定方法
HK40068528A (en) Multiplex pyrosequencing using non-interfering noise canceling polynucleotide identification tags
HK40117503A (en) Single molecule nucleic acid sequence analysis processes and compositions
HK40028998A (en) Single molecule nucleic acid sequence analysis processes and compositions
HK40028998B (en) Single molecule nucleic acid sequence analysis processes and compositions
US20100196893A1 (en) Method for genotyping DNA tandem repeat sequences
HK1257956B (en) Single molecule nucleic acid sequence analysis processes
WO2013140339A1 (fr) Contrôle positif pour pcr
WO2016065182A1 (fr) Systèmes et méthodes de capture d'acides nucléiques
WO2013102061A1 (fr) Amorces et sondes actb

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24824343

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE