WO2011069501A1 - Essai de compétition pour détecter des événements d'hybridation d'oligomères d'acide nucléique - Google Patents

Essai de compétition pour détecter des événements d'hybridation d'oligomères d'acide nucléique Download PDF

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WO2011069501A1
WO2011069501A1 PCT/DE2010/075152 DE2010075152W WO2011069501A1 WO 2011069501 A1 WO2011069501 A1 WO 2011069501A1 DE 2010075152 W DE2010075152 W DE 2010075152W WO 2011069501 A1 WO2011069501 A1 WO 2011069501A1
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nucleic acid
acid oligomers
signal
detection
oligomers
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Gerhard Hartwich
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Friz Biochem Gesellschaft fuer Bioanalytik mbH
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Friz Biochem Gesellschaft fuer Bioanalytik mbH
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a method for detecting nucleic acid oligomer hybridization events.
  • nucleic acids In disease diagnosis, microbiological diagnostics, toxicological testing, genetic research and development, as well as in the agricultural and pharmaceutical sectors, the direct detection of very small amounts of nucleic acids has become indispensable. Increasingly, detection techniques using array technology using so-called DNA chips have become available which enable surface-sensitive detection of nucleic acid oligomer hybridization events. With the help of this sensitive method, nucleic acids can be detected with a very low detection limit.
  • a library of known DNA sequences (“probe oligonucleotides”) is fixed in an ordered grid on a surface, so that the position of each individual DNA sequence is known.
  • target oligonucleotides fragments of active genes whose sequences are complementary to specific probe oligonucleotides on the chip exist in the assay solution, then the target oligonucleotides can be identified by detection of the corresponding on-chip hybridization events.
  • WO 99/51778 A1 describes an electrochemical method for the detection of nucleic acid oligomer hybridization events.
  • the association events are evidenced by the change in the electrochemical properties of the probe molecules associated with the association.
  • WO 2007/143669 A2 discloses a method for the electrochemical detection of nucleic acids by means of probes which are bound to a microarray surface.
  • competitor molecules may be present which compete with the target nucleic acids for binding sites on the immobilized probes.
  • the object of the present invention is to provide a method for detecting nucleic acid oligomer hybridization events which combines high detection sensitivity with easy sample handling.
  • dNTP deoxyribonucleoside triphosphate a mix of dATP
  • dCTP deoxycytidine triphosphate
  • dGTP deoxyguanosine triphosphate
  • dTTP deoxythymidine triphosphate
  • Nucleic acid nucleic acid of unspecified base length e.g.
  • Oligomer Nucleic Acid Octamer A nucleic acid of any type
  • Oligomer equivalent to nucleic acid oligomer Oligomer equivalent to nucleic acid oligomer.
  • Oligonucleotide equivalent to oligomer or nucleic acid oligomer e.g. a DNA, PNA or RNA fragment of unspecified base length.
  • Oligo Abbreviation for oligonucleotide Oligo Abbreviation for oligonucleotide.
  • Nucleic Acid At least two covalently linked nucleotides or at least two covalently linked pyrimidine (e.g., cytosine, thymine or uracil) or purine bases (e.g., adenine or guanine).
  • the term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, e.g. to the sugar-phosphate backbone of the DNA, cDNA or RNA, to a peptide backbone of the PNA or to analogous structures (e.g., phosphoramide, thio-phosphate or dithio-phosphate backbone).
  • An essential feature of a nucleic acid according to the present invention is that it can bind naturally occurring cDNA or RNA sequence-specific.
  • Template nucleic acid molecule equivalent to
  • Sample sample nucleic acid, sample DNA or template.
  • hybridization step also: annealing
  • thermostable polymerase e.g. Taq polymerase (which was originally isolated from a thermophilic organism Thermus aquaticus) whose optimum working temperature is in a range of 70 ° C to 74 ° C and which is also tolerant to temperatures of up to 100 ° C and more.
  • the two single strands hybridize such that the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (in the case of RNA, T is replaced by uracil). Any other base pairing does not form hydrogen bonds, distorts the structure, and is referred to as a "mismatch.”
  • Nucleic acid oligomers hybridize the two single strands, wherein the nucleotide sequence of one strand is complementary to the nucleotide sequence of the other strand, so that the base A (or C) of one strand with the base T (or G) of the other strand hydrogen bonds formed (in RNA T is replaced by uracil).
  • redox-active Designates the property of a unit under certain circumstances to donate electrons to a suitable oxidant or to take up electrons from a suitable reducing agent.
  • linkers are commercially available as alkyl, alkenyl, alkynyl, heteroalkyl, hetero-alkenyl or hetero-alkynyl chain, wherein the chain is derivatized in two places with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partner.
  • the reactive groups may also be photoactivatable, ie the reactive groups are activated only by light of specific or arbitrary wavelength.
  • Nonspecific nt, ie, nt complementary to other bases can also be used as linker / spacer, in particular in the case of the attachment of probe oligos to a surface.
  • the present invention relates to a method of detecting nucleic acid oligomer hybridization events comprising the steps of providing a modified surface, wherein the modification comprises attaching at least one type of probe nucleic acid oligomer, providing a sample with target nucleic acid oligomers, providing a solution having at least one Type of signal nucleic acid oligomers, wherein the signal nucleic acid oligomers are modified with at least one detection label, the signal nucleic acid oligomers have a complementary or substantially complementary to the probe nucleic acid oligomers section and the signal nucleic acid oligomers to the target nucleic acid oligomers complementary or largely complementary section providing a reaction solution for carrying out a nucleic acid amplification, wherein the reaction solution comprises at least nucleotides and at least one type of nucleic acid acid polymerase, mixing and contacting with the modified surface of the solution with signal nucleic acid oligomers, the sample with target nucleic acid oligomers and the reaction solution for
  • the method according to the invention is based on the idea of combining the high sensitivity of a surface-sensitive detection method with a preceding amplification of the target oligonucleotides. Thereby, the limit for the detection of the target nucleic acid oligomers originally present in the sample can be shifted to extremely small concentrations. Ideally, detection of a single target nucleic acid oligomer is possible.
  • a "substantially complementary structure" is understood as meaning sequence sections in which a maximum of 20% of the base pairs form mismatches. the base pairs form mismatches.
  • a "substantially complementary structure” is a sequence segment in which a maximum of 10% of the base pairs form mismatches, and very particularly preferably sequence segments in which a maximum of 5% of the base pairs form mismatches then it is a complementary structure.
  • the method according to the invention enables the detection of nucleic acid oligomer hybridization events with simultaneously proceeding amplification of the target nucleic acid oligomers, wherein probe and signal nucleic acid oligomers have largely complementary sections and at the same time the signal nucleic acid oligomers have a section complementary to the target nucleic acid oligomers.
  • the entire process is carried out in a single reaction vessel and thus represents a "closed tube” application.
  • the amplification of the target nucleic acid oligomers increases their amount and thus their concentration present in the reaction mixture in the course of carrying out the process.
  • concentration ratios of signal nucleic acid oligomers to target nucleic acid oligomers are critical.
  • a method in which surface-sensitive detection and at the same time an amplification are carried out must therefore fulfill certain conditions which allow accurate and error-free detection of nucleic acid oligomer hybridization events.
  • signal nucleic acid oligomers which have a complementary or substantially complementary to the target nucleic acid oligomers portion and at the same time a complementary to the probe nucleic acid oligomers or substantially complementary section, the signal nucleic acid oligomers with both the target nucleic acid oligomers as also hybridize with the probe nucleic acid oligomers. It is a competition assay in which target nucleic acid oligomers and probe nucleic acid oligomers compete for the signal nucleic acid oligomers.
  • Hybridization events are more likely and occur at higher rates when all binding partners are free in solution. Boundary surface reactions, namely, binding events between a free-solvent binding partner and a second binding partner immobilized on a surface are usually slower and less likely to occur. Therefore, hybridization of the free-in-solution target and signal nucleic acid oligomers is much more likely and occurs faster than hybridization of the signal nucleic acid oligomers with the surface-bound probe nucleic acid oligomers. The increase in concentration of the target nucleic acid oligomers due to their amplification enhances this effect so that any increase in the amount of target nucleic acid oligomers makes capture of the signal nucleic acid oligomers more likely.
  • Hybridization events of signal nucleic acid oligomers with the immobilized on the surface probe nucleic acid oligomers are characterized with increasing target nucleic acid oligomer concentration less and less, whereby the measurement signal of the surface-sensitive detection decreases.
  • the probe nucleic acid oligomers may have a greater number of bases than the signal nucleic acid oligomers and comprise a sequence segment having a complementary or substantially complementary structure to the target nucleic acid oligomers.
  • This section which does not have a complementary or substantially complementary structure to a section of the signal nucleic acid oligomers, is referred to in the context of the present invention as a target docking section.
  • the target nucleic acid oligomers In the presence of a target docking section, the target nucleic acid oligomers first hybridize with the target docking section to the probe nucleic acid oligomers and displace in this way particularly fast and efficiently pre-bound signal nucleic acid oligomers.
  • a decrease in the signal intensity over time is detected in the presence of the target being sought and, above all, when the concentration is increased by amplification.
  • the signal nucleic acid oligomers may also comprise a larger number of bases than the probe nucleic acid oligomers and comprise at least one target docking section, wherein the target docking section does not have a complementary or substantially complementary structure to a section of the probe nucleic acid oligomers and wherein the target nucleic acid oligomers in addition to its complementary to the signal nucleic acid oligomers Section have another to the target docking section complementary or largely complementary section.
  • the target nucleic acid oligomers first hybridize with the target docking section to the signal nucleic acid oligomers and displace in this way particularly fast and efficiently previously bound probe nucleic acid oligomers.
  • the amplification of the target nucleic acid oligomers is carried out by a PCR or by an isothermal amplification. If the amplification takes place via a PCR, the provided reaction solution additionally contains at least one type of primer. Isothermal amplification is carried out at a constant temperature, for example at 60 ° C, and depending on the chosen conditions substantially both a linear and an exponential increase of the synthesized nucleic acid molecules is possible.
  • a Rolling Circle Amplification (RCA), a Linear Rolling Circle Amplification (LRCA) or a Primer Generation Rolling Circle Amplification (PG-RCA) are available.
  • RCA Rolling Circle Amplification
  • LRCA Linear Rolling Circle Amplification
  • PG-RCA Primer Generation Rolling Circle Amplification
  • PCR is a fundamental method for molecular biology that essentially replicates DNA molecules and also allows rapid and sensitive detection of nucleic acids.
  • the Polymerase Chain Reaction (PCR) is based on a recurring cycle of three steps occurring at different temperatures, namely denaturation, hybridization and extension. In the denaturation, the reaction mixture is heated to a temperature greater than 90 ° C, preferably 94 ° C to 95 ° C. As a result, the two complementary strands of a double-stranded DNA separate, the DNA is "melted” or denatured and is present in single strands.
  • the denaturation is a very fast process and is usually completed within seconds.
  • the temperature is lowered to a so-called “annealing temperature”.
  • annealing temperature depends on the length and sequence of the primers and can be determined from these properties specifically for each primer.As a rule, the "annealing temperatures” in a range of 55 ° C to 65 ° C, can but also lower or higher depending on the application.
  • the combination of primer and template DNA is most stable if all the bases in the primer are complementary are and will be destabilized by the presence of a so-called mismatch, which in relation to the "annealing temperature” means that in the case of fully complementary primers it can be chosen to be higher than, for example for primers with a mismatch. By choosing the "annealing temperature” more or less stringent hybridization conditions can also be created.
  • the polymerases begin to grow additional complementary nucleotides and thus to synthesize the opposite strand.
  • the link between primers (or newly synthesized backbone) and template is further enhanced with each nucleotide added.
  • the hybridization takes little time and takes place within seconds.
  • the temperature is further increased, preferably to 70 ° C to 74 ° C, which is the ideal Working temperature for the polymerases used in the rule, which grow additional nucleotides to the resulting DNA strands.
  • 70 ° C to 74 ° C which is the ideal Working temperature for the polymerases used in the rule, which grow additional nucleotides to the resulting DNA strands.
  • the loose connections between primers and those template DNA segments that are not completely complementary also break again.
  • the extension step is the step of the PCR, which usually involves the greatest amount of time.
  • the working or reaction rate of the polymerase is time-limited, that is, the shorter the optimal temperature of about 70 ° C to 74 ° C, the shorter the newly synthesized DNA strands remain.
  • a time interval in the range of minutes (from one to several minutes) for DNA extension is selected.
  • Each repetition of the above three steps doubles the number of copied DNA molecules. After 20 cycles, about one million molecules are formed from a single DNA double strand.
  • a special PCR application the so-called real-time PCR allows the detection and quantification of target nucleic acid molecules in real time, ie during the PCR reaction.
  • Real-time PCR is gaining in popularity in the labs, not least because it's fast and precise at the same time.
  • the determination of the amount of DNA at the end of a PCR can namely for many reasons not directly conclusions about the number of originally present molecules because z. B. at the beginning and at the end of the PCR, the conditions for the polymerases are not necessarily optimal and therefore the amplification does not run smoothly over the entire reaction time. Therefore, the quantification at the end of a PCR can be very inaccurate.
  • the detection limits for specific applications can be further optimized.
  • pre-amplifying the sample nucleic acid by means of PCR for example, the number of targets is increased to such an extent that reliable, reliable and accurate detection is possible, for example with DNA chips.
  • the mixture of signal nucleic acid oligomers, target nucleic acid oligomers and reaction solution in contact with the modified surface must undergo thermocyclization to effect amplification.
  • three different temperatures must be applied consecutively, which allow the three steps of a PCR, namely denaturation, hybridization and extension.
  • the denaturation is usually carried out at a temperature of about 95 ° C
  • the temperature for the hybridization is chosen application-specific, but is usually in a range of about 55 ° C to 65 ° C, while the extension at a temperature of about 72 ° C to 74 ° C is performed.
  • At least the signal nucleic acid oligomers have at their 3 " end a modification which prevents the addition of nucleotides by the nucleic acid polymerase
  • a synthesis starting point for the polymerase which could then extend the signal nucleic acid oligomer using the target nucleic acid oligomer as template, by appropriate modification the 3 " ends of the signal nucleic acid oligomers, or by using one or more dideoxynucleotides as last, terminal nucleotides at the 3 " end, ensures that there is no undesirable extension of the signal nucleic acid oligomers during the step of amplification.
  • the probe nucleic acid oligomers have a modification at their 3 " end which prevents attachment of nucleotides by the nucleic acid polymerase, provided that the probe nucleic acid oligomers are bonded to the surface via their 5 " end the 3 'end free of charge.
  • the signal nucleic acid oligomers have at their 3 " end a modification or dideoxynucleotides as last, terminal nucleotides at the 3 " end in order in each case to prevent attachment of nucleotides by the nucleic acid polymerase.
  • the signal nucleic acid oligomers have at least one "non-matchende” base at the 3 ' end, which also prevents extension of the signal nucleic acid oligomers by the polymerase.
  • the method according to the invention for the detection of nucleic acid oligomer hybridization events can therefore be used in a particularly advantageous manner Combination with a device for performing a PCR can be used.
  • the device for carrying out a PCR in this case comprises at least one sample cell with a cavity for receiving a sample and at least three independently adjustable temperature control units, which define three spatially separate temperature zones.
  • At least one means for carrying out a relative movement between the sample cell and the tempering units is provided, wherein the means for carrying out a relative movement is designed and set up such that the sample can be moved through the three spatially separated temperature zones due to the relative movement between the sample cell and the tempering units ,
  • the sample cell comprises a configured and designed for the detection of nucleic acid oligomer hybridization events sensor which is formed in the space provided for receiving the sample cavity of the sample cell.
  • a sensor designed and designed for surface-sensitive detection of nucleic acid oligomer hybridization events.
  • the sensor consists essentially of a modified surface, wherein the modification consists in the connection of at least one type of probe nucleic acid oligomers.
  • surface refers to any support material which is suitable for binding derivatized or non-derivatized probe nucleic acid oligomers covalently or via other specific interactions, directly or after appropriate chemical modification.
  • the solid support may be made of conductive or non-conductive material. Methods for immobilizing nucleic acid oligomers on a surface are known to those skilled in the art.
  • the sensor present in the sample cell provides the modified surface required for the method according to the invention.
  • the device for carrying out a PCR has a fourth temperature control unit which defines a fourth temperature zone spatially separated from the three temperature zones, very particular advantages result.
  • the fourth temperature control unit of the device it becomes possible to subregions of the sample cell, in particular the area in which the sensor is located in one fourth temperature zone, which differs from the temperatures required for carrying out the PCR and at the same time represents an optimal temperature for the surface-sensitive detection of nucleic acid hybridization events, position.
  • the fourth temperature zone is set to a temperature of about 50 ° C. which is favorable for this type of detection.
  • the detection step preferably takes place directly after the denaturation step of the PCR amplification.
  • the temperature of about 95 ° C. prevailing during denaturing all the bonds or hybridizations of nucleic acid molecules are loosened, i. all nucleic acid oligomers are present as a single strand and signal and target nucleic acid oligomers are free in solution.
  • the probability and therefore the speed of hybridization of signal and target nucleic acid oligomers thereby becomes higher than a hybridization of signal and probe nucleic acid oligomers.
  • the signal intensity of the surface-sensitive detection decreases with each PCR cycle.
  • the reaction solution provided for the amplification of the target nucleic acid oligomers additionally contains at least one type of primer by means of PCR.
  • the primers are preferably chosen so that they are complementary to the target nucleic acid oligomers, but not to the probe and signal nucleic acid oligomers.
  • the step of amplification is carried out at least 5 times, in particular at least 10 times and preferably at least 20 times.
  • the detection of the signal nucleic acid oligomers can take place after any number of amplification cycles.
  • the second detection can be carried out, for example, after two or three or four cycles, the next detection of the signal nucleic acid oligomers after a further, ten further or further 20 cycles, the subsequent detection after four further cycles, eight further cycles or twelve further cycles, etc Any conceivable combination of detection steps and amplification cycles is possible.
  • the concentration of the signal nucleic acid oligomers in the produced mixture between 10 "15 mol / l and 10" 5 mol / l, preferably between 10 "13 mol / l and 10" 7 mol / l and more preferably between 10 " 11 mol / l and 10 "7 mol / l.
  • the choice of the concentration ranges and / or the exact adjustment of the concentration of the signal nucleic acid oligomers is such that a stable and uniform measurement over the entire detection period is possible and depends inter alia on which detection label the signal nucleic acid oligomers used.
  • the optimal concentration ranges may differ for signal nucleic acid oligomers with a redox-active detection label and those with a fluorescent label. Likewise, especially with fluorescence-labeled signal nucleic acid oligomers, the number of fluorophores present can influence the optimal concentration.
  • the preferred concentration of signal nucleic acid oligomers depends on the size of the test sites and on the occupancy density with which the probe nucleic acid oligomers are bound to the chip surface.
  • the concentration of the target nucleic acid oligomers in the produced mixture up to 10 is preferably "9 / mol l, preferably up to 10" 11 mol / l, more preferably up to 10 "13 mol / l.
  • the target nucleic acid oligomers undergo a continuous change in the concentration of the target nucleic acid oligomers, with the number of desired amplification cycles also being different depending on the type of application. It makes sense to adjust the number of cycles and the concentration of the target nucleic acid oligomers to one another. The specified limits ensure reliable measurement throughout the entire process.
  • the preferred concentration of the target nucleic acid oligomers depends on the size of the test sites and the occupancy density with which the probe nucleic acid oligomers are bound to the chip surface.
  • the detection of the signal nucleic acid oligomers is effected by a surface-sensitive detection method, since in this case only the signal nucleic acid oligomers bound to the surface are detected. Spectroscopic, electrochemical and electrochemiluminescent methods are preferred in this context.
  • condition are set or measures taken to at least predominantly dissociation of probe nucleic acid oligomers and signal - Lead nucleic acid oligomers.
  • Such dehybridization of the surface-bound double-stranded hybrids substantially returns the modified surface to its original state.
  • a separate dehybridization step is not necessary since the dehybridization takes place automatically during a PCR cycle.
  • At least predominant dissociation of probe nucleic acid oligomers and signal nucleic acid oligomers is meant in the context of the present invention that at least 50% of the surface bonded double-stranded hybrids dissociate, preferably at least 75%, more preferably at least 90% and most preferably at least 98%.
  • TIRF total internal reflection fluorescence
  • SPR surface plasmon resonance
  • the modification of the provided modified surface is the attachment of several types of probe nucleic acid oligomers.
  • the different types of probe nucleic acid oligomers are bound to the modified surface in spatially substantially separated regions.
  • the modified surface preferably has at least 2 spatially substantially separated regions, more preferably at least 4 and in particular at least 12 such spatially substantially separated regions. Most preferably, the modified surface has at least 32, in particular at least 64, most preferably at least 96 spatially substantially separated areas.
  • the provided modified surface has an area of 1 ⁇ m 2 to 1 mm 2 , preferably an area of 10 ⁇ m 2 to 100 ⁇ m 2 , more preferably an area of approximately 50 ⁇ m 2 .
  • the surface area of the modified surface depends, on the one hand, on the number of different types of probe nucleic acid oligomers bound to the modified surface in spatially substantially separated regions. The more spatially substantially separated areas the modified surface should provide, the larger its area must be selected. On the other hand, however, for example, by altering the coverage with probe nucleic acid oligomers, the area of the modified surface can be adjusted within a reasonable range. As a rule, an occupation density of approximately 6 ⁇ 10 12 probe nucleic acid oligomers per cm 2 surface is used. Deviations from this guideline may make larger or smaller areas useful.
  • the present invention also includes a kit for carrying out the method according to the invention.
  • the kit contains at least one modified surface as specified in the present invention and an effective amount of signal nucleic acid oligomers as defined in the present invention.
  • the term "surface” refers to any support material which is suitable for binding derivatized or non-derivatized probe nucleic acid oligomers covalently or via other specific interactions, directly or after appropriate chemical modification.
  • the solid support may be made of conductive or non-conductive material.
  • conductive surface is understood to mean any support having an electrically conductive surface of any thickness, in particular surfaces of platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead , Aluminum and manganese.
  • any doped or non-doped semiconductor surfaces of any thickness can be used. All semiconductors can be used as pure substances or as mixtures. As non-limiting examples are carbon, silicon, germanium, cc at this point Tin, Cu (l) - and Ag (l) halides of any crystal structure called.
  • Also suitable are all binary compounds of any composition and any structure of the elements of groups 14 and 16, the elements of groups 13 and 15, and the elements of groups 15 and 16.
  • ternary compounds of any composition and any structure of the elements of Groups 1 1, 13 and 16 or the elements of groups 12, 13 and 16 are used.
  • the names of the groups of the Periodic Table of the Elements refer to the 1985 IUPAC Recommendation.
  • the preferred material is glass, modified glass or silicon.
  • the modification may e.g. by silanization and leads in all cases to functional groups which are suitable in coupling reactions according to functionalized probe
  • Nucleic acid oligomers bind. This modification includes surface buildup structures using polymers such as dextran polymers which allow for variation of layer thickness and surface finish. Further derivatization possibilities for the final binding of the probe nucleic acid oligomers consist, for example, in the application of a thin (about 10-200 nm) metallization layer, in particular a gold metallization layer, which can additionally be coated with (thiol-functionalized) polymers, in particular dextranes.
  • the glass can also be functionalized with biotin after silanization (eg amino-functionalized glass surface after silanization and coupling of the carboxylic acid biotin via a biotin active ester such as biotin-N-succinimidylester) or alternatively coated with immobilized on dextran lysine or dextran biotin.
  • biotin after silanization eg amino-functionalized glass surface after silanization and coupling of the carboxylic acid biotin via a biotin active ester such as biotin-N-succinimidylester
  • biotinylated glass surfaces thus produced are then treated with avidin or streptavidin and can then be used to attach biotinylated probe nucleic acid oligomers. Binding of nucleic acid oligomers to the surface
  • the probe nucleic acid oligomers may be e.g. covalently bound to the surface via hydroxyl, epoxide, amino or carboxy groups of the support material with thiol, hydroxy, amino or carboxyl groups naturally present on the nucleic acid oligomer or attached by derivatization to the probe nucleic acid oligomer.
  • the probe nucleic acid oligomer can be bound directly or via a linker / spacer to the surface atoms or molecules of a surface.
  • the probe nucleic acid oligomer may be anchored by the methods commonly used in immunoassays, such as e.g.
  • probe nucleic acid oligomers for non-covalent immobilization on avidin or streptavidin-modified surfaces.
  • the chemical modification of the probe nucleic acid oligomers with a surface anchor group can already be introduced in the course of the automated solid-phase synthesis or else in separate reaction steps.
  • the nucleic acid oligomer is linked directly or via a linker / spacer with the surface atoms or molecules of a surface of the type described above. This binding can be carried out in various ways known to those skilled in the art. In this context, reference is made to WO 00/42217 A1.
  • the probe nucleic acid oligomers of the present invention consist of nucleotides in a particular nucleotide sequence (sequence) and are immobilized on a surface.
  • Target nucleic acid oligomers are defined as molecules which interact specifically with the probe nucleic acid oligomers or with the signal nucleic acid oligomers to form a double-stranded hybrid.
  • Target nucleic acid oligomers within the meaning of the present invention are therefore nucleic acid oligomers which act as complex binding partners of the complementary probe nucleic acid oligomer or signal nucleic acid oligomer.
  • Nucleic acid oligomers act.
  • the target nucleic acid oligomers whose Presence is to be detected by the present invention have at least one sequence region whose sequence is complementary or at least substantially complementary to a portion of the probe nucleic acid oligomers or the signal nucleic acid oligomers.
  • the target nucleic acid oligomers are present in the sample either as a single strand (ss) or as a double strand (ds).
  • the target nucleic acid oligomers are at least partially present as a single strand. This can e.g. be achieved by ds-target nucleic acid oligomers (thermally or by other measures known in the art) are dehybridized or that care is taken in the preparation of the target nucleic acid oligomers that the target nucleic acid oligomers z.T. exist as single strands. This is achieved for example by asymmetric PCR.
  • a nucleic acid oligomer or ns-oligomer is a compound of at least two covalently linked nucleotides or of at least two covalently linked pyrimidine (eg cytosine, thymine or uracil) or purine bases (eg adenine or guanine), preferably a DNA , RNA or PNA fragment.
  • pyrimidine eg cytosine, thymine or uracil
  • purine bases eg adenine or guanine
  • nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as the sugar-phosphate backbone of the DNA, cDNA or RNA, to a peptide backbone of the PNA, or to analogous backbone structures, such as a thio-phosphate, a dithio-phosphate or a phosphoramide backbone.
  • An essential feature of a nucleic acid according to the present invention is the sequence-specific binding of naturally occurring DNA, or RNA or derived (transcribed or amplified) structures such as cDNA or amplified cDNA or amplified RNA (aRNA). Detection label / marker (marker molecule)
  • the signal nucleic acid oligomers are provided by derivatization with one or more detectable labels.
  • This label allows the detection of the complexing events between the signal nucleic acid oligomer and the surface-bound probe nucleic acid oligomers.
  • the label can directly or as in the case of enzyme-catalyzed reactions indirectly provide a detection signal.
  • Preferred detection labels are fluorophores and redox-active substances.
  • fluorescent dyes such as e.g. Texas red, rhodamine dyes, fluorescein, etc. (see Molecular Probes catalog).
  • redox molecules are used as labels.
  • redox label transition metal complexes in particular those of copper, iron, ruthenium, osmium or titanium with ligands such as pyridine, 4,7-dimethylphenanthroline, 9,10-phenanthrenquinonediimine, porphyrins and substituted porphyrin derivatives can be used.
  • riboflavin of quinones such as pyrroloquinolinoquinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone or derivatives thereof, of metallocenes and metallocene derivatives such as ferrocenes and ferrocene derivatives, cobaltocenes and cobaltocene derivatives, of porphyrins, methylene blue, daunomycin, dopamine derivatives, hydroquinone Derivatives (para- or ortho-dihydroxy-benzene derivatives, para- or ortho-dihydroxy-anthraquinone derivatives, para- or ortho-dihydroxy-naphthoquinone derivatives) and similar compounds possible.
  • quinones such as pyrroloquinolinoquinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone or derivatives thereof
  • Indirect labels can also be used in the processes according to the invention.
  • the term "indirect labels” is understood to mean those in which the actually detectable form of the label is first formed via an enzyme-catalyzed reaction The detectable form of the label can then be detected on the surface Examples of such indirect labels are known to those skilled in the literature
  • alkaline phosphatase (AP) in connection with the substrate p-aminophenyl phosphate is known here as an indirect marker bound to the signal nucleic acid oligomer
  • an electrochemical detection of the signal nucleic acid oligomer can be effected by adding p-aminophenyl phosphate at the time of detection.
  • the electrochemically inactive p-aminophenyl phosphate serves as a substrate of the enzyme AP and is converted into p-aminophenol. After diffusion to a conductive surface, p-aminophenol can now be detected electrochemically, since this form of the substrate (ie after conversion at the AP) is electrochemically active.
  • AP can also be used for chromogenic detection (eg with 5-bromo-4-chloro-3-indoxyl phosphate in conjunction with nitroblue tetrazolium chloride).
  • Detection methods allow the distinction between marker molecules associated with a surface and those dissolved in the supernatant. Detection methods are electrochemical, spectroscopic and electrochemiluminescent methods. (i) Surface Sensitive Electrochemical Detection
  • the kinetics of the electrochemical processes can in principle be used to distinguish between redox-active detection labels adsorbed on a surface and dissolved in the supernatant.
  • Surface adsorbed detection labels are generally more rapidly electrochemically reacted (e.g., oxidized or reduced) as the redox-active, volume phase detection label since the latter must first diffuse to the (electrode) surface prior to electrochemical conversion.
  • electrochemical surface-sensitive methods include cyclic voltammetry, amperometry and chronocoulometry.
  • the method of chronocoulometry makes it possible to distinguish near-surface redox-active components of (identical) redox-active components in the bulk phase and is, for example, in Steel, AB, Herne, TM and Tarlov MJ: Electrochemical Quantitation of DNA Immobilized on Gold, Analytical Chemistry, 1998, Vol. 70, 4670-4677 and references cited therein.
  • the use of chronocoulometry in a displacement assay for the detection of nucleic acid oligomer hybridization events is described in detail in WO 03/018834 A2, which is hereby incorporated by reference.
  • the optical measurement method used to detect fluorescently labeled signal nucleic acid oligomers is total internal reflection fluorescence (TIRF, see Sutherland and Dahne, 1987, J. Immunol., Meth., 74, 253-265).
  • TIRF total internal reflection fluorescence
  • Fluorescent molecules located near the interface between a solid waveguide medium, typically glass, and a liquid medium or immobilized on the liquid-facing surface of the waveguide medium may be excited by the evanescent field protruding from the waveguide and emit detectable fluorescent light.
  • Repressed or supernatant-dissolved fluorescently labeled complexing agents are not detected by the evanescent field (or only insofar as they are in the range of the penetration depth of the evanescent field) and thus provide (almost) no contribution to the measured signal.
  • the penetration depth of the evanescent field is typically 100 to 200 nm, but can be increased to several 100 nm by a thin metallization layer (about 10 to 200 nm), in particular a gold metallization layer.
  • the layer thickness of the probe-modified carrier surface is adapted to the penetration depth of the evanescent field, for example by correspondingly long probe nucleic acid oligomers, by immobilization of the probe nucleic acid oligomers via a correspondingly long linker between the surface and probe oligonucleotide, by coupling the carboxylic acid biotin (via a biotin active such as biotin-N-succinimidylester) on amino-derivatized surfaces and coupling of avidin or streptavidin to the biotinylated surfaces thus produced with subsequent attachment of biotinylated probe nucleic acid oli
  • 1A a schematic representation of the measuring principle of the detection of 1 B nucleic acid oligomer hybridization events according to the method of the invention
  • 2 shows a schematic representation of a sample cell with integrated sensor
  • FIG. 3 shows a perspective view of a device for carrying out a PCR reaction with simultaneous detection of nucleic acid oligomer hybridization events
  • 5A a plot of the measurement results of the detection of 5B, 5C, nucleic acid oligomer hybridization events at 24 test sites 5D during a PCR (Multiplex Real-Time PCR).
  • FIGS. 1A, 1B schematically show the measuring principle of the detection of nucleic acid oligomer hybridization events during a PCR, namely an electrically detected real-time PCR, wherein in FIG. 1A the state at the beginning or before the start of the PCR and in FIG B is the state of an advanced PCR reaction outlined.
  • signal nucleic acid oligomers 2 with covalently bonded ferrocenes 3 are added to a PCR reaction batch containing target nucleic acid oligomers 1.
  • the signal nucleic acid oligomers 2 are complementary, on the one hand, to the probe nucleic acid oligomers 5 immobilized on a test site 4 of a DNA chip and, on the other hand, to the target nucleic acid oligomers 1.
  • the covalently bonded ferrocenes 3 of the signal nucleic acid oligomers 2 can be used for electrochemical detection of the signal nucleic acid oligomers 2 bound / hybridized to the test sites 4 (electrodes) since ferrocene is reversibly oxidizable and reducible.
  • test sites 4 electrodes
  • ferrocene is reversibly oxidizable and reducible.
  • the free-in-solution signal nucleic acid oligomers 2 are present in a concentration which produces no measurable additional signal at the test site 4 or they can on the basis of their time characteristic - free signal nucleic acid oligomers 2 must first diffuse to the test site 4 and can then be oxidized or - be discriminated by bound at the test site 4, hybridized with probe nucleic acid oligomers signal nucleic acid oligomers. Thermal dehybridization (heating of the DNA chip and the adjacent solution to 95 ° C.) leads to diffusion of all signal nucleic acid oligomers 2 bound to the test site. Test site 4 is restored in its original form with free probe nucleic acid oligomers 5.
  • This condition exists at least for the period in which the temperature after thermal dehybridization is above the melting temperature of the hybrid of probe and signal nucleic acid oligomers 25.
  • a lowering of the temperature at the test site 4 of the DNA chip to or below the melting temperature of the hybrid 25 allows a new measurement.
  • the number of target nucleic acid oligomers 1 increases exponentially.
  • appropriate temperature control melting of the PCR products, lowering the temperature to a range below the melting temperature of a hybrid of signal and target nucleic acid oligomers 12, then optionally further lowering the temperature to a range below the melting temperature of the hybrid Signal and probe nucleic acid oligomers 25
  • the concentration of the free signal nucleic acid oligomers 2 decreases markedly, which leads to a slower hybridization of the signal nucleic acid oligomers 2 at the test site 4 and to a reduced degree of hybridization between probe and signal nucleic acid oligomers 25.
  • a signal nucleic acid oligomer 2 can be destroyed (separation of label and oligosequence), which, in addition to the consumption of free signal nucleic acid oligomers 2 leads to a permanent consumption of the signal nucleic acid oligomers 2 by the (correct) amplification process and thus the signal at the test site 4 of the DNA chip even more strongly influenced by the hybridization to the target nucleic acid oligomers 12.
  • the method according to the invention for detecting nucleic acid oligomer hybridization events can be used in combination with a device 30 for carrying out a PCR.
  • a specific embodiment of the device 30 is shown in FIG.
  • the device 30 for carrying out the PCR comprises a sample cell 10 with a cavity for receiving the sample and three independently adjustable temperature control units, which define three spatially separate temperature zones. For the sake of clarity, only the temperature control units 7a and 7c are shown in FIG.
  • a means R for carrying out a relative movement between the sample cell and the temperature control units is provided, wherein the means R for performing a relative movement is designed and arranged so that the sample due to the relative movement between the sample cell 10 and the Temperature units is moved through the three spatially separated temperature zones.
  • sample cell 10 is a means for performing rotational movements, namely a rotatable axis R.
  • a specific embodiment of the sample cell 10 is shown in FIG.
  • the sample cell 10 has an approximate diameter of 20 mm and a thickness of 1 mm.
  • the existing in the sample cell 10 cavity 8 has a depth of about 0.5 mm and two filling holes 1 1 for charging with the PCR reaction mixture.
  • the sample cell 10 comprises a sensor 20 set up and designed for the detection of nucleic acid oligomer hybridization events, namely a DNA chip which is formed in the cavity 8 of the sample cell 10.
  • the DNA chip 20 present in the sample cell 10 provides the modified surface required for the method according to the invention and has a sensor field 20a which is in contact with the reaction solution via a bridge channel 8.1.
  • the DNA chip 20 has a plated-through hole, so that the electrical contacting of the DNA chip, which is necessary for the measurement value recording, can be accomplished from outside via spring contacts 9 (see FIG. 2) which are formed in the rotatable axis R.
  • the three independent tempering units of the device 30 each have a temperature-controllable block 6 of a good thermal conductivity material, in the specific example of aluminum.
  • a temperature-controllable block 6 of a good thermal conductivity material, in the specific example of aluminum.
  • PID controller platinum resistance for temperature measurement and suitable control electronics (PID controller)
  • the temperature-controllable blocks 6 are brought to the temperature necessary for the PCR reaction.
  • the first temperature control unit 7a defines a first temperature zone of 95.degree. C.
  • the second temperature control unit 7c defines a second temperature zone of 55.degree. C.
  • the third temperature control unit (not shown) defines a third temperature zone of 72.degree.
  • Each temperature-controllable block 6 contains a gap 13 into which the sample cell 10 is introduced.
  • the device 30 has a fourth temperature control unit 7d, which defines a fourth temperature zone spatially separated from the other temperature zones.
  • the rotatable axis R also represents the fourth temperature control unit 7d, wherein two cylindrical, temperature-controllable blocks 6 are arranged above and below the sample cell 10 in the region of the DNA chip.
  • the fourth temperature-regulating unit 7d of the device makes it possible to position partial regions of the sample cell, in particular the region in which the sensor is located, in a fourth temperature zone, which provides an optimum temperature for the surface-sensitive detection of nucleic acid hybridization events.
  • the area in which the sample cell is heated to a certain temperature can be set.
  • a 2: 1: 1 ratio of the 72 ° C range to the 95 ° C and 55 ° C ranges was chosen.
  • CMOS-based DNA chip for electrochemical detection of hybridization between probe and signal nucleic acid oligomers is described, for example, in Augustyniak, M .; Paul, C; Brederlow, R .; Persike, N .; Hartwich, G .; Schmitt-Landsiedel, D .; Thewes, R. (2006), Solid State Circuits, 2006 IEEE International Conference Digest of Technical Papers, 59-68 A 24x16 CMOS-Based Chronoculometric DNA Microarray.
  • PCR polymerase is added and the solution filled into the sample cell 10 as shown in FIG.
  • the reaction mixture present in the sample cell 10 is heated in the device 30, as shown in FIG. 2, to 95 ° C. for 3 minutes, then cooled and a first measurement is carried out on the integrated DNA chip 20.
  • the DNA chip 20 carries at one of the provided test sites probe nucleic acid oligomers which are complementary to the signal nucleic acid oligomers and at another test site probes which are complementary to the control signal nucleic acid oligomers.
  • This first measurement takes place at 62 ° C, about 4 ° C below the melting temperature of the hybrid of probe and signal nucleic acid oligomers under the conditions in the reaction solution and well below the melting temperature of the hybrid of target and signal nucleic acid oligomers (the signal Nucleic acid oligomer has more matching bases to the target nucleic acid oligomer than to the probe nucleic acid oligomer).
  • the melting temperature of the hybrid of control probe nucleic acid oligomer and control signal nucleic acid oligomer under the conditions in U.S. Pat Reaction solution is identical to the melting temperature of the hybrid of probe and signal nucleic acid oligomers.
  • the result of this normalization measurement is shown in FIG. 4A.
  • the measurement at the control test site is marked with K.
  • the curves shown in Figures 4A, 4B, 4C represent the integral oxidation current of the ferrocene labels from the signal nucleic acid oligomers hybridized to the probe nucleic acid oligomers versus time and thus reflect the degree of hybridization or hybridization efficiency, respectively.
  • the hybridization efficiency in turn depends on the concentration of free signal nucleic acid oligomers and thus indirectly on the concentration or the amount of target nucleic acid oligomers.
  • the temperature-controllable blocks of the device 30 are set to 96 ° C (melting), 55 ° C (annealing) and 72 ° C (elongation) and the sample cell 10 is rotated at a speed of 2 Revolutions per minute (ie 2 cycles per minute) in the temperature-controlled blocks.
  • the nearly centrically aligned sensor array 20a of the DNA chip 20 is held at about 50 ° C. via a further temperature-controllable block, in order then to be heated first to 95 ° C. before re-measurement, in order to obtain the signal nucleic acid oligomers which have undergone a previous measurement are hybridized to the probe nucleic acid oligomers, dehybrid ensue of these again.
  • FIGS. 4B and 4C show results of further measurements after 10 or 20 cycles of the PCR. A slight, approximately 20% decrease in the efficiency of hybridization (of the maximum signal of the electrochemical measurement) is observed at the control test site (indicated by K in the graphs), whereas the hybridization efficiency at the actual detection site increases with increasing efficiency As expected, the amplification rate of the target nucleic acid oligomers decreases (approximately by a factor of 6).
  • FIG. 5 shows the result of such a closed tube multiplex real-time PCR with electrical detection at 24 test points of a DNA chip.
  • FIG. 5 shows two measurement curves for each test site, namely those of the first measurement before the start of the PCR (5.1) and that of the measurement after 25 cycles of the PCR (5.2). The curves represent the current flow (indicated in nA) at the respective test site.
  • the two measurements are essentially identical for almost all test sites, ie the corresponding subtypes (indicated in the respective graphs with the addition “low” or “high”) were not present.
  • the subtypes HPV_6low (see FIG. 5C), HPV_16high and HPV_18high (see FIG. 5D) could be detected; at the test site for HPV_1 1 low (see FIG. 5C) the slight decrease after 25 cycles to a suboptimal test site is per se low signal strength attributed.

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Abstract

L'invention concerne un procédé de détection d'événements d'hybridation d'oligomères d'acide nucléique comprenant les étapes: préparation d'une surface modifiée, la modification consistant à lier au moins un type d'oligomères d'acide nucléique sondes, préparation d'un échantillon avec des oligomères d'acide nucléique cibles, préparation d'une solution avec au moins un type d'oligomères d'acide nucléique signaux, les oligomères d'acide nucléique signaux étant modifiés par au moins une étiquette de détection, les oligomères d'acide nucléique signaux possédant un segment complémentaire ou largement complémentaire des oligomères d'acide nucléique sondes et les oligomères d'acide nucléique signaux possédant un segment complémentaire ou largement complémentaire des oligomères d'acide nucléique cibles, préparation d'une solution de réaction pour effectuer une amplification de l'acide nucléique, la solution de réaction contenant au moins des nucléotides et au moins un type de polymérase d'acide nucléique, mélange et mise en contact avec la surface modifiée de la solution avec des oligomères d'acide nucléique, de l'échantillon avec des oligomères d'acide nucléique cibles et de la solution de réaction pour effectuer une amplification de l'acide nucléique, les oligomères d'acide nucléiques signaux étant présents dans le mélange produit à une concentration de 10-17 moles/l à 10-3 moles/l et les oligomères d'acide nucléique cibles étant présents dans le mélange produit à une concentration de 10-7 moles/l, première détection des oligomères d'acide nucléique signaux par une méthode sensible à la surface, amplification des oligomères d'acide nucléique cibles au moyen d'une amplification de l'acide nucléique, deuxième détection des oligomères d'acide nucléiques signaux par une méthode sensible à la surface et comparaison des valeurs obtenues lors de la première détection des oligomères d'acide nucléique signaux avec les valeurs obtenues lors de la deuxième détection des oligomères d'acide nucléique signaux.
PCT/DE2010/075152 2009-12-07 2010-12-03 Essai de compétition pour détecter des événements d'hybridation d'oligomères d'acide nucléique Ceased WO2011069501A1 (fr)

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EP3802866A4 (fr) * 2018-06-05 2022-03-09 Valorisation-Recherche, Limited Partnership Systèmes cinétiquement programmés et réactions de détection moléculaire

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DE102020106865A1 (de) 2020-03-12 2021-09-16 Analytik Jena Gmbh Anordnung und Verfahren zur PCR mit mehrkanaliger Fluoreszenzmessung für räumlich verteilte Proben

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