WO2022120914A1 - Procédé de mesure de la longueur d'un produit d'amplification d'une ou de plusieurs molécules d'acide nucléique dans un échantillon - Google Patents

Procédé de mesure de la longueur d'un produit d'amplification d'une ou de plusieurs molécules d'acide nucléique dans un échantillon Download PDF

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WO2022120914A1
WO2022120914A1 PCT/CN2020/137054 CN2020137054W WO2022120914A1 WO 2022120914 A1 WO2022120914 A1 WO 2022120914A1 CN 2020137054 W CN2020137054 W CN 2020137054W WO 2022120914 A1 WO2022120914 A1 WO 2022120914A1
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nucleic acid
polymerase
group
detection probe
thermus
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PCT/CN2020/137054
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Chinese (zh)
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宋娜杰
倪润芳
黄丽婷
郭婷婷
王亚芳
何昌华
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厦门致善生物科技股份有限公司
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

Definitions

  • the present invention provides a method for detecting the length of amplification products of one or more nucleic acid molecules in a sample using melting curve analysis.
  • the traditional method for detecting the length of nucleic acid molecules mainly includes agarose gel electrophoresis, which is an electrophoresis method using agar or agarose as a support medium.
  • agarose gel electrophoresis is an electrophoresis method using agar or agarose as a support medium.
  • agarose gels with larger pore sizes can generally be used for electrophoresis separation.
  • Agarose gel has a network structure, substance molecules will be resisted when passing through, and macromolecular substances will receive great resistance when surging. Therefore, in gel electrophoresis, the separation of charged particles depends not only on the nature and number of net charges, but also on the Also depends on molecular size.
  • DNA molecules have charge effect and molecular sieve effect when they migrate in agarose gel. DNA molecules are negatively charged in solution above the isoelectric point and move towards the positive pole in an electric field. Due to the repetitive nature of the sugar-phosphate backbone, double-stranded DNA with the same number of nucleotides has almost the same amount of net charge, so they can move toward the positive pole at the same rate. Nucleic acid fragment lengths can be determined using agarose electrophoresis.
  • agarose gel electrophoresis alkaline agarose gel method
  • traditional detection methods eg, agarose gel electrophoresis, alkaline agarose gel method
  • steps such as gel preparation, electrophoresis, and gelation are required.
  • requirements for electrophoresis time are more strict.
  • the operation of the alkaline agarose gel method is complicated, and steps such as gel preparation, electrophoresis, neutralization, staining, and gelation are required. And it is time-consuming, and the electrophoresis process only takes several hours.
  • amplification product refers to an amplified nucleic acid produced by amplifying a nucleic acid template.
  • polymerase also known as polymerase
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA-dependent DNA polymerases DNA-dependent DNA polymerases
  • DNA-dependent RNA polymerases DNA-dependent RNA polymerases
  • RNA-dependent RNA polymerases RNA-dependent RNA polymerases.
  • the first two are DNA polymerases, and the latter two are RNA polymerases.
  • DNA polymerase refers to an enzyme that uses nucleic acid strands as templates to synthesize DNA strands.
  • DNA polymerases use existing DNA or RNA as templates to synthesize DNA.
  • the DNA polymerase may be a naturally occurring DNA polymerase, or a variant or fragment of a natural enzyme having the above-mentioned activities.
  • the term "RNA polymerase” refers to an enzyme that uses nucleic acid strands as templates to synthesize RNA strands.
  • RNA polymerases use existing DNA or RNA as templates to synthesize RNA.
  • the RNA polymerase may be a naturally occurring RNA polymerase, or a variant or fragment of a natural enzyme having the above-mentioned activities.
  • reverse transcriptase refers to an enzyme capable of synthesizing or replicating RNA into complementary DNA or cDNA. Reverse transcription is the process of copying an RNA template into DNA.
  • the reverse transcriptase may be a naturally occurring RNA polymerase, or a variant or fragment that retains the above activities.
  • forward and reverse are used only for convenience in describing and distinguishing two primers in a primer pair; they are relative, does not have a special meaning.
  • a sequence in a detection probe that is capable of specifically hybridizing to a specified region of the nucleic acid molecule is also referred to as a "targeting sequence” or “target-specific sequence”
  • target sequence and “target” “Specific sequence” refers to a sequence capable of selectively/specifically hybridizing or annealing to a target nucleic acid sequence under conditions that permit hybridization, annealing, or amplification of the nucleic acid, which comprises a sequence complementary to the target nucleic acid sequence.
  • targeting sequence and “target-specific sequence” have the same meaning and are used interchangeably. It is readily understood that a targeting sequence or target-specific sequence is specific for a target nucleic acid sequence.
  • a targeting sequence or target-specific sequence only hybridizes or anneals to a specific target nucleic acid sequence, and not to other nucleic acid sequences, under conditions that allow nucleic acid hybridization, annealing, or amplification.
  • the term “complementary” means that two nucleic acid sequences are capable of forming hydrogen bonds between each other according to the principles of base pairing (Waston-Crick principle), and thereby forming duplexes.
  • the term “complementary” includes “substantially complementary” and “completely complementary”.
  • the term “completely complementary” means that every base in one nucleic acid sequence is capable of pairing with bases in another nucleic acid strand without mismatches or gaps.
  • the term "substantially complementary” means that a majority of bases in one nucleic acid sequence are capable of pairing with bases in the other nucleic acid strand, which allows for mismatches or gaps (eg, one or mismatches or gaps of several nucleotides).
  • two nucleic acid sequences that are "complementary” eg, substantially complementary or fully complementary
  • non-complementary means that two nucleic acid sequences cannot hybridize or anneal under conditions that permit hybridization, annealing, or amplification of the nucleic acids to form a duplex.
  • not perfectly complementary means that bases in one nucleic acid sequence cannot perfectly pair with bases in another nucleic acid strand, at least one mismatch or gap exists.
  • hybridization and “annealing” mean the process by which complementary single-stranded nucleic acid molecules form a double-stranded nucleic acid.
  • hybridization and “annealing” have the same meaning and are used interchangeably.
  • two nucleic acid sequences that are completely complementary or substantially complementary can hybridize or anneal.
  • the complementarity required for hybridization or annealing of two nucleic acid sequences depends on the hybridization conditions used, in particular the temperature.
  • PCR reaction has the meaning commonly understood by those skilled in the art, which refers to a reaction (polymerase chain reaction) that amplifies a target nucleic acid using a nucleic acid polymerase and primers.
  • the term "detection probe” refers to an oligonucleotide labeled with a reporter group and a quencher group.
  • the quencher group When the probe is not hybridized to other sequences, the quencher group is positioned to absorb the signal of the quenched reporter group (eg, the quencher group is located adjacent to the reporter group), thereby absorbing or quenching the reporter group signal sent. In this case, the probe does not emit a signal.
  • the quencher group is located in a position that cannot absorb or quench the signal of the reporter group (eg, the quencher group is located away from the reporter group), so that it cannot absorb or quench the signal of the reporter group. Quench the signal from the reporter group. In this case, the probe emits a signal.
  • melting curve analysis has the meaning commonly understood by those skilled in the art and refers to the analysis of the presence or identity of a double-stranded nucleic acid molecule by determining the melting curve of the double-stranded nucleic acid molecule. method, which is commonly used to assess the dissociation characteristics of double-stranded nucleic acid molecules during heating. Methods for performing melting curve analysis are well known to those skilled in the art (see, e.g., The Journal of Molecular Diagnostics 2009, 11(2):93-101). In this application, the terms “melting curve analysis” and “melting analysis” have the same meaning and are used interchangeably.
  • melting curve analysis can be performed by using a self-quenching probe labeled with a reporter group and a quencher group.
  • probes are capable of forming duplexes with their complementary sequences through base pairing.
  • the reporter group such as a fluorophore
  • the quencher group on the probe are separated from each other, and the quencher group cannot absorb the signal (such as a fluorescent signal) emitted by the reporter group.
  • the strongest signal eg fluorescent signal
  • the two strands of the duplex begin to dissociate (ie, the probe gradually dissociates from its complementary sequence), and the dissociated probe assumes a single-stranded free coil state.
  • the reporter group eg, fluorophore
  • the quencher group on the dissociated probe are in close proximity to each other, whereby the signal (eg, fluorescence signal) emitted by the reporter group (eg, fluorophore) is absorbed by the quenching group. Therefore, as the temperature increases, the detected signal (eg, the fluorescence signal) gradually becomes weaker.
  • the two strands of the duplex are completely dissociated, all probes are in a single-stranded free coil state.
  • the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) on the probe is absorbed by the quencher group.
  • the signal (eg, fluorescent signal) emitted by the reporter group (eg, fluorophore) is substantially undetectable. Therefore, by detecting the signal (such as a fluorescent signal) emitted by the duplex containing the probe during the heating or cooling process, the hybridization and dissociation process of the probe and its complementary sequence can be observed, and the signal intensity changes with the temperature change. changing curve.
  • a curve with the change rate of the signal intensity as the ordinate and the temperature as the abscissa ie, the melting curve of the duplex
  • the peak in the melting curve is the melting peak
  • the corresponding temperature is the melting point (T m ) of the duplex.
  • T m melting point
  • melting curve analysis can be performed by using detection probes labeled with reporter and quencher groups.
  • the detection principle is the same as above.
  • the present application uses detection probes to perform melting curve analysis on amplification products, thereby realizing the detection of nucleic acid molecule amplification products.
  • the application provides a method for detecting the length of an amplification product of one or more nucleic acid molecules in a sample, the method comprising:
  • the detection probe (b) providing at least one detection probe labeled with a reporter group and a quencher group, wherein the reporter group can emit a signal, and the quencher group can absorb or quench the signal emitted by the reporter group; and the detection probe emits a signal when hybridized to its complementary sequence that is different from the signal emitted when it is not hybridized to its complementary sequence; under conditions that allow nucleic acid hybridization or annealing Under the following conditions, the detection probe can specifically hybridize to a designated region of the nucleic acid molecule;
  • step (c) the nucleic acid molecule is mixed with the polymerase and the primer set, and amplified, and then, after the amplification, a detection probe is added to the in the product of step (b), and performing melting curve analysis; or, in step (b), mixing the nucleic acid molecule with the polymerase, the primer set and the detection probe, and performing amplification , and then, after the end of amplification, perform melting curve analysis.
  • the method further comprises, prior to step (c), providing deoxynucleoside triphosphates (dNTPs), water, a solution containing an ion (eg, Mg 2+ ), a single-stranded DNA binding protein, or any combination thereof.
  • dNTPs deoxynucleoside triphosphates
  • water a solution containing an ion (eg, Mg 2+ ), a single-stranded DNA binding protein, or any combination thereof.
  • the sample comprises or is DNA, RNA, or any combination thereof.
  • the nucleic acid molecule is selected from DNA, RNA, or any combination thereof.
  • the amplification product of the nucleic acid molecule is selected from DNA, RNA, or any combination thereof. In certain embodiments, the amplification product of the nucleic acid molecule is DNA.
  • the sample is derived from eukaryotes (eg, animals, plants, fungi), prokaryotes (eg, bacteria, actinomycetes), viruses, bacteriophages, or any combination thereof.
  • eukaryotes eg, animals, plants, fungi
  • prokaryotes eg, bacteria, actinomycetes
  • viruses e.g., viruses, bacteriophages, or any combination thereof.
  • the polymerase is selected from a DNA polymerase, an RNA polymerase, or any combination thereof.
  • the polymerase is a DNA polymerase obtained from a bacterium selected from the group consisting of: Thermus aquaticus (Taq), Thermus thermophiles (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranildanii,Thermus caldophllus,Thermus chliarophilus,Thermus flavus,Thermus igniterrae,Thermus lacteus,Thermus oshimai,Thermus ruber,Thermus rubens,Thermus scotoductus,Thermus silvanus,Thermus thermophllus,Thermotoga maritima,Thermotoga neapolitana,Thermosipho africanus,Thermococcus litoralis,Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermus bacter
  • the polymerase is a DNA polymerase selected from the group consisting of Bst DNA polymerase, T7 DNA polymerase, phi29 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, Pfu DNA polymerase, vent DNA polymerase, or any combination thereof.
  • the polymerase is a DNA polymerase including reverse transcriptase.
  • the polymerase is a reverse transcriptase selected from the group consisting of MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, or any combination thereof.
  • step (a) of the method for each nucleic acid molecule, at least one pair of primer sets is provided, the primer sets comprising at least one forward primer and at least one reverse primer.
  • the forward primer and reverse primer each independently comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.
  • the detection probe has a nucleotide sequence that is complementary (eg, fully complementary) to a specified region of the nucleic acid molecule.
  • the designated region is a designated distance from the region to which the primer hybridizes (eg, 100nt, 200nt, 300nt, 400nt, 500nt, 800nt, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt, 5000nt, or other designation the distance).
  • step (b) of the method at least one detection probe is provided for each nucleic acid molecule amplification product (eg, 1, 2, 3, 4 are provided , 5, 6, 7, 8, 9, 10, or more detection probes).
  • At least two detection probes are provided for each nucleic acid molecule's amplification product, wherein the first detection probe is capable of interacting with The first region of the nucleic acid molecule is hybridized, and the distance between the first region and the region where the forward primer is hybridized is the first distance, and the second detection probe can hybridize with the second region of the nucleic acid molecule, and the second region is hybridized with the forward primer.
  • the distance of the area is the second distance, and the second distance is greater than the first distance.
  • the melting curve analysis According to whether there is a corresponding melting peak and/or melting point (T m ) in the melting curve analysis, it can be determined whether there is an amplification product complementary to the corresponding detection probe. increase product.
  • the amplification of the nucleic acid molecule is completed, and only the first melting peak corresponding to the duplex formed by the first detection probe is detected by the melting curve analysis, it can be determined that the length of the amplified product is greater than the first distance and less than the second distance.
  • the melting curve analysis is performed to detect the first melting peak corresponding to the duplex formed by the first detection probe
  • the first melting peak corresponding to the duplex formed by the second detection probe is also detected.
  • there are two melting peaks it can be determined that the length of the amplified product is greater than the second distance.
  • each detection probe is independently capable of specifically hybridizing to a different designated region of the nucleic acid molecule under conditions that permit hybridization or annealing of the nucleic acid.
  • each detection probe independently has a nucleotide sequence that is complementary (eg, fully complementary) to a different designated region of the nucleic acid molecule.
  • the length of the amplification product is determined according to whether there is a corresponding melting peak and/or the size of the melting point (T m ) in the melting curve analysis.
  • the melting points (T m values) of different duplexes formed by combining different detection probes with their complementary sequences can be calculated in advance. Therefore, according to whether there is a corresponding melting peak and/or melting point (T m ) in the melting curve analysis, it can be determined whether there is an amplification product complementary to the corresponding detection probe, and then, according to the specific hybridization of the detection probe region, the length of the amplified product can be determined.
  • the detection probes are designed such that the duplexes formed by each detection probe have different Tm values from each other. Therefore, by detecting the presence or absence of melting peaks of different duplex melting points (T m values) in the melting curve analysis, it is possible to determine whether there is an amplification product complementary to the corresponding detection probe, and then determine the length of the amplification product .
  • different detection probes can be labeled with the same or different reporter groups (eg, fluorophores).
  • detection probes are designed such that different detection probes are labeled with different reporter groups (eg, fluorophores).
  • the different reporter groups can be detected in different detection channels.
  • the duplexes formed by different detection probes may have the same or different Tm values.
  • detection probes are designed such that duplexes formed by different detection probes have different melting points ( Tm values), and different detection probes are labeled with different reporter groups (eg, fluorophores) .
  • Tm values melting points
  • reporter groups eg, fluorophores
  • each detection probe has a different melting point ( Tm ) between the double-stranded hybrid formed by the amplification product of the nucleic acid molecule.
  • the melting point ( Tm ) between the detection probe and the double-stranded hybrid formed by the amplification product of the nucleic acid molecule differs by 1°C (eg, 1°C, 2°C, 3°C) )above.
  • the detection probes each independently comprise or consist of naturally occurring nucleotides (eg, deoxyribonucleotides or ribonucleotides), modified nucleotides, non-natural nucleosides acid (eg, peptide nucleic acid (PNA) or locked nucleic acid), or any combination thereof.
  • naturally occurring nucleotides eg, deoxyribonucleotides or ribonucleotides
  • modified nucleotides eg, non-natural nucleosides acid (eg, peptide nucleic acid (PNA) or locked nucleic acid
  • the detection probes are each independently 15-1000nt in length, eg, 15-20nt, 20-30nt, 30-40nt, 40-50nt, 50-60nt, 60-70nt, 70-80nt , 80-90nt, 90-100nt, 100-200nt, 200-300nt, 300-400nt, 400-500nt, 500-600nt, 600-700nt, 700-800nt, 800-900nt, 900-1000nt.
  • the detection probes each independently have a 3'-OH terminus; alternatively, the 3'-terminus of the detection probes is blocked; Add a chemical moiety (eg, biotin or alkyl) to the 3'-OH of the acid by removing the 3'-OH of the last nucleotide of the detection probe, or by replacing the last nucleotide with a double deoxynucleotides, thereby blocking the 3'-end of the detection probe.
  • a chemical moiety eg, biotin or alkyl
  • step (d) the product of step (c) is gradually heated or cooled and the signal emitted by the reporter group on each detection probe is monitored in real time, thereby obtaining each A curve of the signal intensity of the reporter group as a function of temperature; the curve is then derived to obtain a melting curve for the product of step (d).
  • the detection probes are each independently labeled with a reporter group at their 5' end or upstream and a quencher group at their 3' end or downstream, or labeled at their 3' end or downstream.
  • the reporter group is labeled and the quencher group is labeled at the 5' end or upstream.
  • the reporter group and the quencher group are separated by a distance of 10-80 nt or more.
  • the reporter groups in the detection probe are each independently a fluorophore (eg, ALEX-350, FAM, VIC, TET, CAL Gold 540, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 590, ROX, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red 635, Quasar 670, CY3, CY5, CY5.5, Quasar 705); and a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
  • a quenching group is a molecule or group capable of absorbing/quenching the fluorescence (eg, DABCYL, BHQ (eg, BHQ-1 or BHQ-2), ECLIPSE, and/or TAMRA).
  • the detection probes each independently have the same or different reporter groups. In certain embodiments, the detection probes each independently have the same or different quencher groups.
  • the detection probes are each independently resistant to nuclease activity (eg, 5' nuclease activity, eg, 5' to 3' exonuclease activity); eg, the detection probes
  • the backbone of the needle contains modifications that resist nuclease activity, such as phosphorothioate linkages, alkyl phosphotriester linkages, aryl phosphotriester linkages, alkyl phosphonate linkages, arylphosphonate linkages, hydrophosphates bond, alkyl phosphoramidate bond, aryl phosphoramidate bond, 2'-O-aminopropyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-O- Butyl modification, and 1-(4'-thio-PD-ribofuranosyl) modification.
  • the detection probes are each independently linear, or have a hairpin structure.
  • sequence of the detection probe is selected from SEQ ID Nos: 6, 7, 8, 9, or any combination thereof.
  • the detection method of the present application is different from the traditional detection method in the past, and the length of the amplification product of one or more nucleic acid molecules can be detected by using melting curve analysis.
  • the method of the present application can simultaneously determine the lengths of amplification products of multiple nucleic acid molecules.
  • the operation is simple and the steps are simple, and the reaction time and the detection time are saved at the same time.
  • Figure 1 shows the results of melting curve analysis using detection probe 1 to detect the length of fragment 1; wherein, the solid black line represents the detection result of fragment 1, and the solid gray line represents the detection result of the negative control.
  • Figure 2 shows the results of melting curve analysis using detection probe 2 to detect the length of fragment 2; wherein, the solid black line represents the detection result of fragment 2, and the solid gray line represents the detection result of the negative control.
  • Figure 3 shows the results of melting curve analysis using detection probe 3 to detect the length of fragment 3; wherein, the solid black line represents the detection result of fragment 3, and the solid gray line represents the detection result of the negative control.
  • Figure 4 shows the results of melting curve analysis using detection probe 4 to detect the length of fragment 4; wherein, the solid black line represents the detection result of fragment 4, and the solid gray line represents the detection result of the negative control.
  • Figure 5 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 5.
  • Figure 6 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 6.
  • Figure 7 shows the results of melting curve analysis using detection probes 1-4 to detect the length of fragment 7.
  • Figure 8 shows the results of melting curve analysis using detection probes 1-4 to detect the length of Fragment 8.
  • ⁇ DNA purchased from Life technologies (Shanghai) was used as the template to construct fragments of different lengths.
  • the PCR method and corresponding primers are used for amplification, wherein the length of the amplified fragment 1 is 2024 bp, the length of the amplified fragment 2 is 3036 bp, the length of the amplified fragment 3 is 4026 bp, and the length of the amplified fragment 4 is is 5044bp, and the primers used are shown in Table 1.
  • the enzyme used for PCR amplification was 2 ⁇ TaKaRa Taq TM HS Perfect Mix (purchased from TaKaRa). The specific reaction systems are shown in Tables 2 to 6.
  • probes 1-4 are designed to bind to the amplified product, wherein probe 1 can bind to the 1152nt to 1189nt nucleotide sequence of the amplified product, and probe 2 Can bind to the 2108 nt to 2134 of the nucleotide sequence of the amplification product, probe 3 can bind to the 3137 nt to 3167 nt of the nucleotide sequence of the amplification product, and probe 4 can bind to the 4246 nt to 4274 nt of the nucleotide sequence of the amplification product .
  • the lengths of fragments 1-4 were determined by melting curve analysis, respectively.
  • the probe is hybridized with it at a lower temperature, and the double-strand formed by the probe and the amplification product is calculated by gradually increasing the temperature and detecting its fluorescence signal.
  • the melting temperature (T m ) corresponding to the body is used to indicate whether there is a corresponding detection target by the presence or absence of a melting peak at the corresponding temperature of the probe.
  • the probes used in this example are shown in Table 7, and the fluorescent quantitative PCR reaction program is shown in Table 8.
  • the detection system used was 25 ⁇ L: 2.5 ⁇ L of 10x PCR buffer, 1.5 ⁇ L of MgCl 2 (25 mM), 2 ⁇ L of 5 ⁇ M probe (probe 1-4), 12.5 ⁇ L of amplified fragment (fragment 1-4), 6.5 ⁇ L of RNase Free Water.
  • the formula of 10 ⁇ PCR buffer is: (NH 4 ) 2 SO 4 21.142 g, Tris 81.164 g, Tween-20 1.0 mL, pH 8.8.
  • the measurement results of the lengths of fragments 1-4 are shown in Figures 1 to 4, respectively.
  • the solid gray line is the negative control, and the solid black line is the nucleic acid fragment detected.
  • the results prove that the length of the nucleic acid fragment can be detected by the method of the present invention, and the detection result is accurate.
  • ⁇ DNA purchased from Life technologies (Shanghai)
  • the ⁇ DNA was amplified by the PCR method and the primers shown in Table 9, and four amplifications with different product lengths were carried out, wherein the length of fragment 5 was 1463 bp, the length of fragment 6 was 2875 bp, and the length of fragment 7 was 3362 bp , the length of fragment 8 is 4528bp.
  • 2xTaKaRa Taq TM HS Perfect Mix purchased from TaKaRa was used for PCR amplification, and the specific reaction systems are shown in Table 10 to Table 13.
  • the assays were carried out respectively, and the detection system used in this example is shown in Table 14.
  • the formula of 10x PCR buffer is: (NH 4 ) 2 SO 4 21.142 g, Tris 81.164 g, Tween-20 1.0 mL, pH 8.8.

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Abstract

L'invention concerne un procédé de mesure de la longueur d'un produit d'amplification d'une ou de plusieurs molécules d'acide nucléique dans un échantillon. Le procédé utilise une technologie d'analyse de courbe de fusion. Le procédé est pratique à utiliser et simple quant à ses étapes, et réduit également le temps de mesure.
PCT/CN2020/137054 2020-12-08 2020-12-17 Procédé de mesure de la longueur d'un produit d'amplification d'une ou de plusieurs molécules d'acide nucléique dans un échantillon WO2022120914A1 (fr)

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CN202011420931.9 2020-12-08
CN202011420931.9A CN114606298A (zh) 2020-12-08 2020-12-08 一种检测样品中一种或多种核酸分子扩增产物长度的方法

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