Method for isothermal amplification of nucleic acid molecules

Abstract

The invention relates to the field of nucleic acid amplification, in particular to a method for isothermal amplification of nucleic acid molecules. The invention provides a method for isothermal amplification of nucleic acid molecules. The method comprises the following steps: mixing nucleic acid to be amplified with helicase to obtain single-stranded nucleic acid; mixing the single-stranded nucleic acid with the single-stranded binding protein and the primer enzyme to obtain oligonucleotide; under the condition that dNTP exists, oligonucleotide and DNA polymerase are mixed, and an extension product is obtained; repeating the steps for one or more times on the extension product to obtain the sequence of the nucleic acid to be amplified. The HPA disclosed by the invention is an isothermal amplification method which comprises the following steps of: unfastening double strands of a template by utilizing helicase, generating a primer by utilizing primer enzyme, and then amplifying by utilizing DNA (Deoxyribose Nucleic Acid) polymerase. According to the method, no additional primer needs to be added, non-specific amplification products such as primer dimers are prevented from being generated, high-temperature denaturation is also not needed, and the reaction can be completed at a constant temperature. Whole genome amplification can be carried out on an original template, and the DNA concentration of the original template is improved, so that downstream detection is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid amplification, and in particular to a method for isothermal amplification of nucleic acid molecules. Background Art

[0002] Polymerase chain reaction (PCR) is a method for rapidly amplifying specific DNA fragments in vitro. Since Mullis's invention of PCR in 1985, PCR technology has undergone rapid innovation and has played a significant role in the molecular diagnosis of genetic diseases, pathogens, oncogenes, and other areas, as well as in forensic identification. However, conventional PCR technology has drawbacks such as high instrumentation costs, prolonged amplification times, and the need for temperature fluctuations. Since the 21st century, several nucleic acid amplification technologies have been developed that are simpler and more convenient in terms of both instrumentation requirements and practical operation, demonstrating promising application prospects in nucleic acid testing. Common isothermal amplification techniques include: Loop-Mediated Isothermal Amplification (LAMP), Multiple Displacement Amplification (MDA), Nucleic Acid Sequences Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Helicase-Dependent Amplification (HDA), etc.

[0003] Primer extension preamplification (PEP) is one of the earliest methods for whole-genome amplification. It is a nucleic acid amplification method developed based on PCR technology. Its main principle is to use 15-base random primers (N15, theoretically with 415 combinations) to anneal for a long time at a low annealing temperature of 37°C, then slowly raise the temperature to 55°C for a long primer extension, and repeat this process for multiple cycles. However, because the PEP method uses random primers and less stringent PCR cycle parameters, it may lead to uneven amplification results and random mutations. Most nucleic acid amplification methods based on PCR technology have shortcomings due to the inherent disadvantages of temperature variation, which may lead to problems such as cumbersome operation and primer-primer interactions.

[0004] Multiple displacement amplification (MDA) is a method based on isothermal nucleic acid amplification. Its basic principle is to use a thiolated hexanucleotide random primer (N6) to randomly anneal to the genome under constant temperature. This is followed by strand displacement amplification by the strong strand displacement activity of phi29 DNA polymerase. The resulting single-stranded sequence can then anneal and extend with the random primer at will, forming a hyperbranched amplification structure. Due to the strong processivity of phi29 DNA polymerase, it can continuously synthesize products up to 50–100 kb in length. Because MDA is an isothermal nucleic acid amplification method, nonspecific amplification may occur, meaning that a large amount of product is produced even in the absence of template.

[0005] Helicase-dependent amplification (HDA) is an in vitro isothermal amplification technique that mimics the DNA replication mechanism in animals. It utilizes components such as helicase, single-stranded DNA binding protein (SSB), and strand-displacing polymerase to replace the thermal cycling process of denaturation, annealing, and extension in PCR. The principle is that the helicase unwinds the double-stranded DNA structure, while the SSB binds to the single strand, stabilizing it. Primers then bind to the single-stranded target sequence, and strand-displacing polymerase promotes the synthesis of new double-stranded DNA. This newly synthesized double-stranded DNA serves as a template for the next round of amplification, repeating the unwinding-amplification steps. The HAD method has a simple amplification process, closely resembling the denaturation, annealing, and extension steps of PCR. However, the requirement for primers limits its application to whole-genome amplification, and the addition of primers inevitably leads to the formation of primer dimers or other products.

[0006] Helicases are a class of enzymes that unwind hydrogen bonds, using ATP hydrolysis to provide energy to unwind DNA strands. DNA helicases are ubiquitous in nature and are found in a variety of prokaryotes, eukaryotes, bacteriophages, and viruses. Most organisms also encode multiple helicases, such as Escherichia coli, which encodes at least 12 different helicases. DNA helicases are important motor proteins that unwind double-stranded DNA to produce transient single-stranded DNA intermediates required for replication, recombination, and repair. In these processes, duplex DNA is unwound by DNA helicases, which act by binding to and hydrolyzing nucleoside 5'-triphosphates (NTPs) to break the hydrogen bonds between complementary base pairs in duplex DNA.

[0007] Primase is used to guide the synthesis of RNA primers, which in turn guide DNA polymerase-mediated DNA chain synthesis. There are two types of primases: DnaG-type primases and archaeal and eukaryotic primases (AEPs). Primpols (primase-polymerases) belong to the AEP type of primases and exhibit DNA polymerase and DNA primase activities. They do not require NTPs and typically associate with helicases to form a replication initiation complex.

[0008] Although existing isothermal amplification methods can also amplify the entire genome, they still have limitations. Summary of the Invention

[0009] In light of this, the present invention provides a method for isothermal amplification of nucleic acid molecules. HPA (helicase-primase amplification) refers to an isothermal amplification method that uses a helicase to unwind a double-stranded template, a primase to generate primers, and then amplification using a DNA polymerase. This method does not require the addition of additional primers, thus avoiding the generation of nonspecific amplification products such as primer dimers. It also does not require high-temperature denaturation, and the reaction can be completed at a constant temperature. It can perform whole-genome amplification of the original template and increase the DNA concentration of the original template, thereby facilitating downstream detection.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a method for isothermal amplification of nucleic acid molecules, comprising the following steps:

[0012] S1: Mix the nucleic acid to be amplified with helicase to obtain single-stranded nucleic acid;

[0013] S2: the single-stranded nucleic acid is mixed with a single-stranded binding protein and a primer enzyme to obtain an oligonucleotide;

[0014] S3: In the presence of dNTPs, the oligonucleotide is mixed with DNA polymerase to obtain an extension product;

[0015] S4: The extension product repeats S1 to S3 above once or multiple times to obtain the sequence of the nucleic acid to be amplified.

[0016] In some embodiments of the present invention, the above method does not include the step of adding primers and / or primer sets.

[0017] In some embodiments of the present invention, the mixing in the above method S1 further comprises the step of mixing with ATP.

[0018] In some embodiments of the present invention, in the above method, the concentration of ATP is 100 mM.

[0019] In some embodiments of the present invention, in the above method, the helicase comprises any one of: UvrD helicase from Thermusthermophilus, UvrD helicase from Thermoanaerobacter tengcongensis, and PcrA helicase from Geobacillus stearothermophilus.

[0020] In some embodiments of the present invention, in the above method, the helicase is UvrD helicase derived from Thermoanaerobacter tengcongensis.

[0021] In some embodiments of the present invention, in the above method, the final concentration of the helicase is 10 ng / μL.

[0022] In some embodiments of the present invention, in the above method, the primer enzyme includes: DnaG type primer enzyme or AEP type primer enzyme.

[0023] In some embodiments of the present invention, in the above method, the primase is an AEP-type primase.

[0024] In some embodiments of the present invention, in the above method, the primase is primpol primase derived from Thermus thermophilus.

[0025] In some embodiments of the present invention, in the above method, the final concentration of the primer enzyme is 2.5 ng / μL.

[0026] In some embodiments of the present invention, in the above method, the single-chain binding protein is derived from Escherichiacoli or Thermus thermophilus.

[0027] In some embodiments of the present invention, in the above method, the final concentration of the single-chain binding protein is 0.4-5 mg / mL.

[0028] In some embodiments of the present invention, in the above method, the DNA polymerase includes: phi29 DNA polymerase or Bst3.0 DNA polymerase.

[0029] In some embodiments of the present invention, in the above method, the DNA polymerase is: phi29 DNA polymerase.

[0030] In some embodiments of the present invention, in the above method, the isothermal amplification time is 40 to 60 minutes and the temperature is 42 to 65°C.

[0031] In some embodiments of the present invention, in the above method, the isothermal amplification time is 60 minutes and the temperature is 42°C.

[0032] In some embodiments of the present invention, in the above method, the isothermal amplification time is 40 minutes and the temperature is 65°C.

[0033] The present invention also provides an enzyme composition comprising: a helicase, a primase, a DNA polymerase and a single-strand binding protein.

[0034] In some embodiments of the present invention, in the above enzyme composition, the volume ratio of the helicase, the primase, the DNA polymerase and the single-stranded binding protein is: (1-1.5): (0.2-1): (1-1.5): (0.7-9.55).

[0035] In some embodiments of the present invention, in the enzyme composition, the volume ratio of the helicase, the primase, the DNA polymerase and the single-stranded binding protein is: 1:0.5:1:0.7.

[0036] The present invention also provides a reaction reagent comprising: the above enzyme composition, dNTPs, ATP, DTT and a buffer;

[0037] The reaction reagents do not include: primers and / or primer sets.

[0038] In some embodiments of the present invention, the reaction reagents further include: DMSO and ammonium sulfate.

[0039] In some embodiments of the present invention, in the above reaction reagent, the final concentration of ATP is 100 mM.

[0040] In some embodiments of the present invention, in the above reaction reagents, the final concentration of DTT is 1M.

[0041] In some embodiments of the present invention, in the above reaction reagents, the final concentration of ammonium sulfate is 4M.

[0042] In some embodiments of the present invention, in the above reaction reagents, the buffer solution is obtained by mixing 200 mM Tris-acetate, 500 mM potassium acetate, 100 mM magnesium acetate and 1 mg / mL BSA pH 7.9.

[0043] In some embodiments of the present invention, the reaction reagents include: 2 μL 10× buffer, 0.4 μL dNTPs (25 mM each), 1 μL 100 mM ATP, 0.2 μL 1 M DTT, 0.5 μL 0.1 mg / mL primer, 1 μL DNA polymerase, 1 μL helicase (0.2 mg / mL), 0.7 μL 5 mg / mL single-stranded binding protein, 1 μL DMSO and 0.2 μL 4 M ammonium sulfate solution.

[0044] The present invention provides a product comprising: the above enzyme composition and / or the above reaction reagent and an acceptable auxiliary agent.

[0045] The present invention also provides the use of the above enzyme composition, the above reaction reagent and / or the above product in isothermal amplification.

[0046] The present invention provides a method for isothermal amplification of nucleic acid molecules, comprising the following steps:

[0047] S1: Mix the nucleic acid to be amplified with helicase to obtain single-stranded nucleic acid;

[0048] S2: mixing the single-stranded nucleic acid with a single-stranded binding protein and a primer enzyme to obtain an oligonucleotide;

[0049] S3: In the presence of dNTPs, the oligonucleotide is mixed with DNA polymerase to obtain an extension product;

[0050] S4: The extension product repeats S1 to S3 above once or multiple times to obtain the sequence of the nucleic acid to be amplified.

[0051] HPA (helicase-primase amplification) is an isothermal amplification method that uses a helicase to unwind a double-stranded template, a primase to generate primers, and then amplification using a DNA polymerase. This method does not require the addition of additional primers, thus avoiding the generation of nonspecific amplification products such as primer dimers. It also does not require high-temperature denaturation and can complete the reaction at a constant temperature, significantly shortening the reaction time compared to existing technologies. It can perform whole-genome amplification of the original template and increase the DNA concentration of the original template, thereby facilitating downstream detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0053] Figure 1Figure 1 illustrates the reaction principle of the isothermal amplification method provided by the present invention; wherein: a shows helicase catalyzing the unwinding of a double-stranded DNA structure to form a single-stranded nucleic acid; b shows primase binding to the single strand unwound by the helicase; c shows primase initiating primer synthesis along the 5'→3' direction of the single strand; d shows the primer generated by the binding of a strand-displacing polymerase continuously extending along the 5'→3' direction; e shows primers generated at multiple sites, and the strand-displacing enzyme amplifying all sites simultaneously; wherein: the orange sphere represents the helicase, the green pentagon represents the primase, and the red triangle represents the strand-displacing polymerase;

[0054] Figure 2 Shows the gel image of HPA amplified plasmid agarose detection;

[0055] Figure 3 Shows the gel image of HPA amplified 9948 genome agarose detection;

[0056] Figure 4 The capillary electrophoresis gel images of the 9948 genome amplified by HPA are shown; the upper part shows the capillary electrophoresis image after HPA (assuming that HPA has no amplification effect and the amount of template added for PCR is 0.004 ng); the lower part shows the unamplified 9948 control (the amount of template added for PCR is 0.1 ng);

[0057] Figure 5 Figure 2 shows the fluorescence quantitative PCR images of HPA-amplified M2 DNA; from left to right: after HPA (assuming no amplification, the amount of template added to qPCR was 0.4 ng, and the recovery volume was 50 μL), before HPA (the amount of template added to qPCR was 2 ng), and NTC;

[0058] Figure 6 Figure 2 shows capillary electrophoresis patterns of HPA-treated template amplified using a forensic kit at the human genomic STR locus 25A. The top graph shows the capillary electrophoresis pattern after HPA (assuming HPA has no amplification effect and the amount of template added for PCR is 1.4 pg), while the bottom graph shows the unamplified 9948 control (the amount of template added for PCR is 10 pg).

[0059] Figure 7 Indicates that no helicase or ATP was added to the HPA; wherein, from top to bottom, the recovered product after amplification without adding ATP, the recovered product after amplification in the normal HPA system, the recovered product after amplification without adding helicase, and the capillary electrophoresis image after 25A amplification of 35pg 9948 genomes without amplification;

[0060] Figure 8 The capillary electrophoresis diagrams of different helicases after HPA reaction are shown; wherein: from top to bottom are the capillary electrophoresis diagrams of some sites of Tth-UvrD, Tte-UvrD, and pCRA helicase;

[0061] Figure 9The capillary electrophoresis diagram of HPA using phi 29 DNA polymerase is shown; wherein: the upper part shows the capillary electrophoresis diagram after HPA using phi 29 (assuming that HPA has no amplification effect and the amount of template added for PCR is 1.4 pg); the lower part shows the unamplified 9948 control (the amount of template added for PCR is 10 pg). DETAILED DESCRIPTION

[0062] The invention discloses a method for isothermal amplification of nucleic acid molecules.

[0063] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0064] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0065] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0066] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0067] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specified value or range.

[0068] The present invention provides a method for isothermal amplification of nucleic acid molecules, comprising the following steps: in a single reaction system and at a constant temperature, a nucleic acid helicase breaks hydrogen bonds in the nucleic acid to be amplified, thereby unwinding the double-stranded nucleic acid into a single strand; a single-strand binding protein binds to the single strand, thereby converting the nucleic acid into a single-stranded state; a primer enzyme then binds to the single strand to synthesize an oligonucleotide chain; and a DNA polymerase is used to extend the oligonucleotide in the presence of dNTPs to form a new double-stranded nucleic acid. These steps are repeated cyclically to ultimately obtain a large amount of the desired nucleic acid to be amplified.

[0069] Furthermore, the method includes mixing helicase, primase, single-strand binding protein, DNA polymerase, reaction buffer, etc.

[0070] Furthermore, the sources of the helicase include prokaryotes, eukaryotes, phages and viruses.

[0071] Furthermore, the helicase is one or a combination of Geobacillus stearothermophilus PcrA helicase or Thermoanaerobacter tengcongensis Tte UvrD helicase.

[0072] Furthermore, the sources of the primase include DnaG type primase and archaeal and eukaryotic primase (AEP) types.

[0073] Furthermore, the primer enzyme belongs to the AEP type primer enzyme, and exhibits DNA polymerase activity and DNA initiation enzyme activity, and does not require the participation of NTPs.

[0074] Furthermore, the primer enzyme is Tthprimpol primer enzyme-polymerase (Tth-primpol) derived from Thermus thermophilus.

[0075] Furthermore, the sources of the single-stranded binding protein include bacteriophage and bacteria.

[0076] Furthermore, the single-stranded binding protein is one or a combination of Escherichia coli single-stranded binding protein (E. coli SSB) or Thermus thermophilus SSB (Tth-SSB).

[0077] Furthermore, the DNA polymerase should have a strand displacement function.

[0078] Furthermore, the DNA polymerase is Bacillus stearothermophilus DNA polymerase I large fragment (Bst3.0 DNA polymerase).

[0079] In Examples 1 to 7 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0080] The present invention will be further described below in conjunction with the embodiments:

[0081] Example 1 Amplification of the plasmid after enzyme digestion

[0082] The digested plasmid was amplified using the HPA method, and the amplification was verified by agarose gel electrophoresis. First, 10× HPA reaction buffer (200mM Tris-acetate, 500mM potassium acetate, 100mM magnesium acetate, 1mg / mL BSA pH 7.9) was prepared. The HPA reaction system was prepared as follows: 2μL 10× HPA reaction buffer, 0.4μL dNTPs (25mM each), 1μL 100mM ATP, 0.2μL 1M DTT, 0.9μL 5mg / mL Ecoli SSB, 1μL Bst3.0 DNA polymerase, 1μL TteUvrD helicase (0.2mg / mL), and 1μL DMSO.

[0083] Using the pTrc99A plasmid (15 ng / μL) recovered by EcoRI digestion as a template, the following reagents were added to the HPA reaction system, and finally water was added to the final volume to 20 μL. Four sets of reactions were performed:

[0084] a) Add 0.2 μL of 0.1 mg / mL Tth-primpol and 1 μL of template;

[0085] b) 0.5 μL 0.1 mg / mL Tth-primpol and 1 μL template;

[0086] c) 1 μL 0.1 mg / mL Tth-primpol and 1 μL template;

[0087] d) 0.5 μL of 0.1 mg / mL Tth-primpol, 1 μL of template, and 0.2 μL of 4 M ammonium sulfate solution.

[0088] The four systems were amplified at 65°C for 40 min, and 2 μL was then run on a 1% agarose gel. Quantitative detection was performed using Qubit. Figure 2 As shown, HPA amplification of the digested plasmid yielded numerous bands, consistent with the results from whole-genome amplification. Using ammonium sulfate increased the proportion of large fragments. Under certain conditions, increasing the amount of primers reduced the proportion of large fragments. The Qubit quantification results in Table 1 indicate that HPA amplification increased the total amplification from 15 ng to approximately 1890 ng, a 126-fold increase.

[0089] Figure 2Channel 1 shows the HPA results with 20 ng of primer, channel 2 shows the HPA results with 50 ng of primer, channel 3 shows the HPA results with 100 ng of primer, and channel 4 shows the HPA results with 50 ng of primer added to ammonium sulfate. The gel images show that channel 4 performs better.

[0090] Table 1 Qubit quantitative data of HPA amplified plasmids

[0091] Serial number Concentration ng / μL 1 63.6 2 62.4 3 62.2 4 74.4

[0092] Example 2 Amplification of Human Genome 9948

[0093] Human genome 9948 was amplified using the HPA method and recovered using a recovery kit. The recovered product was used as a template for amplification verification using the Xinhai Biotech Human Genome STR Locus 25A Forensic Kit and capillary electrophoresis. The HPA reaction system consisted of: 3 μL 10× HPA reaction buffer, 0.6 μL 25 mM dNTPs, 1.5 μL 100 mM ATP, 0.3 μL 1 M DTT, 0.75 μL 0.1 mg / mL Tth-primpol, 1.5 μL Bst3.0 DNA polymerase, and 1.5 μL TteUvrD helicase (0.2 mg / mL). The HPA system was supplemented with different volumes of 1 μL, 5 μL, and 9.55 μL of 0.4 mg / mL Tth-SSB; 0 μL and 1 μL of DMSO; and 0 μL and 0.3 μL of 4 M ammonium sulfate solution. Then, 10 μL of 0.3 ng / μL human genome 9948 template was added, and the reaction was incubated at 65°C for 40 minutes. After the reaction, the product was recovered using a gel recovery kit, observed on an agarose gel, and quantified using a Qubit assay. The recovered product was then amplified using a human genome STR locus 25A forensic kit and analyzed by capillary electrophoresis.

[0094] From the electrophoresis results Figure 3 Look, HPA can successfully amplify 9948, combined with Figure 4 As you can see, the HPA-amplified product can be amplified using the Human Genome STR Locus 25A Forensic Kit, and the peak is higher. Selecting channel 2 conditions allows for better template amplification. Compared to the template before HPA amplification, the amplified template exhibits a higher peak height in capillary electrophoresis.

[0095] Figure 3Channel 1 shows the HPA result of 1 μL Tth-SSB, channel 2 shows the HPA result of 5 μL Tth-SSB, channel 3 shows the HPA result of 9.55 μL Tth-SSB, channel 4 shows the HPA result of 5 μL Tth-SSB without DMSO, channel 5 shows the HPA result of 5 μL Tth-SSB without ammonium sulfate, and channel 6 shows the HPA result of 5 μL Tth-SSB without DMSO or ammonium sulfate.

[0096] Table 2 HPA amplified human genome 9948 Qubit quantitative data

[0097] Serial number Concentration ng / μL 1 95.2 2 107.2 3 106.8 4 74.4 5 65.6 6 65.4

[0098] Figure 4 The top is the capillary electrophoresis diagram after HPA (assuming that HPA has no amplification effect and the amount of template added for PCR is 0.004 ng), and the bottom is the unamplified 9948 control (the amount of template added for PCR is 0.1 ng).

[0099] Example 3 Detection of amplification efficiency using fluorescent quantitative PCR

[0100] The M2 DNA template was amplified using the HPA method and recovered using a recovery kit. The recovered product was used as a template for amplification verification using detection reagents and qPCR validation. The HPA reaction system consisted of: 2 μL 10× HPA reaction buffer, 0.4 μL dNTPs (25 mM each), 1 μL 100 mM ATP, 0.2 μL 1 M DTT, 0.5 μL 0.1 mg / mL Tth-primpol, 1 μL Bst3.0 DNA polymerase, 1 μL Tte UvrD helicase (0.2 mg / mL), 1 μL 0.4 mg / mL Tth-SSB, 1 μL DMSO, and 0.2 μL 4 M ammonium sulfate solution. 10 μL of 1 ng / μL template was added to the reaction system, and the reaction was incubated at 65°C for 40 min. After the reaction, the product was recovered using a gel recovery kit and amplified using detection reagents, and qPCR validation was performed. From the fluorescence quantitative PCR test, it can be seen that the original template concentration is improved after HPA amplification, and the Ct value of fluorescence quantitative PCR is advanced.

[0101] Example 4 Detection of the effect of pre-amplification by capillary electrophoresis

[0102] Low-copy number templates were amplified using the HPA method and recovered using a recovery kit. The recovered product was used as a template for amplification verification using the Xinhai Biotech Human Genomic STR Locus 25A Forensic Kit and capillary electrophoresis. The HPA reaction system consisted of: 2 μL 10× HPA reaction buffer, 0.4 μL 25 mM dNTPs, 1 μL 100 mM ATP, 0.2 μL 1 M DTT, 0.5 μL 0.1 mg / mL Tth-primpol, 1 μL Bst3.0 DNA polymerase, 1 μL Tte UvrD helicase (0.2 mg / mL), 0.7 μL 5 mg / mL E. coli SSB, 1 μL DMSO, and 0.2 μL 4 M ammonium sulfate solution. 7 μL of 5 pg / μL template was added to the reaction system, and the reaction was incubated at 65°C for 40 min. After the reaction is completed, the gel recovery kit is used to recover the product, and then the recovered product is used as a template to amplify the human genome STR site 25A forensic kit and run capillary electrophoresis for analysis. Figure 6 It can be seen that when amplifying 5 pg / μL of the genome, the template treated with HPA can significantly increase the peak height detected by the human genome ST R site 25A forensic kit.

[0103] Example 5 Verification of the role of helicase in HPA

[0104] Helicase requires ATP energy to open double-stranded DNA. Removing ATP or helicase from the HPA reaction will verify whether it has an effect on HPA.

[0105] HPA amplification was performed using the low-copy number 9948 genome as a template. The product was recovered using a recovery kit. The recovered product was then amplified and validated using the Xinhai Biotech Human Genome STR Locus 25A Forensic Kit and capillary electrophoresis. The HPA reaction system consisted of: 2 μL 10× HPA Reaction Buffer, 0.4 μL 25 mM dNTPs, 0.2 μL 1 M DTT, 0.5 μL 0.1 mg / mL Tth-primpol, 1 μL Bst3.0 DNA Polymerase, 0.7 μL 5 mg / mL E. col i SSB, 1 μL DMSO, and 0.2 μL 4 M ammonium sulfate solution.

[0106] After preparing the mix, divide the mixture into 3 tubes. One tube is supplemented with 1 μL of 100mM ATP, another tube is supplemented with 1 μL of Tte UvrD helicase (0.2mg / mL), and the last tube is supplemented with ATP and helicase. 7 μL of 5pg / μL template is added to the reaction system and the reaction is carried out at 65℃ for 40 minutes. After the reaction is completed, the product is recovered using a gel recovery kit. The recovered product is then used as a template for amplification using the human genomic STR locus 25A forensic kit and analyzed by capillary electrophoresis. Figure 7 As can be seen in the figure, when amplifying a 5 pg / μL genome, no amplification was observed without the addition of helicase. However, the addition of ATP alone improved amplification, with the addition of both significantly increasing the peak height detected by the human genome STR locus 25A forensic kit. HPA-treated templates with the addition of both helicase and ATP significantly increased the peak height detected by the human genome STR locus 25A forensic kit.

[0107] Figure 7 The middle is the 25A electrophoresis diagram of HPA with single deletion of helicase, ATP and control.

[0108] Example 6 HPA reaction using different helicases

[0109] In the HPA reaction, helicase and polymerase are the main factors limiting the HPA reaction, affecting the efficiency of the amplified fragment, the length of the fragment, etc.

[0110] Use different helicases for the HPA reaction to select the optimal helicase. Amplify the low-copy number 9948 genome and recover it using a recovery kit. The recovered product is used as a template for amplification verification using the Xinhai Biotech Human Genome STR Locus 25A Forensic Kit and capillary electrophoresis. Prepare the HPA reaction system: 2 μL 10× HPA Reaction Buffer, 0.4 μL dNTPs (25 mM each), 1 μL 100 mM ATP, 0.2 μL 1 M DTT, 0.5 μL 0.1 mg / mL Tth-primpol, 1 μL Bst3.0 DNA Polymerase, 0.7 μL 5 mg / mL E. coli SSB, 1 μL DMSO, and 0.2 μL 4 M ammonium sulfate solution. 1 μL Tte-UvrD helicase (0.2 mg / mL), 1 μL Tth-UvrD helicase (0.2 mg / mL), and 1 μL pCRA helicase (0.2 mg / mL) were added to the reaction system. 7 μL 5 pg / μL human genome 9948 template was then added and the reaction was carried out at 65°C for 40 minutes. After the reaction, the product was recovered using a gel recovery kit. The recovered product was then amplified using a human genome STR site 25A forensic kit using the recovered product as a template and analyzed by capillary electrophoresis. Figure 8It can be seen that when amplifying a 5pg / μL genome, using Tte-UvrD helicase to perform HPA treatment on the genome can obtain more balanced and less biased results. The pCRA helicase is second only to the effect, and the Tth-UvrD helicase is the poorest.

[0111] Figure 8 From top to bottom in the figure are the capillary electrophoresis images of some sites of Tth-UvrD, Tte-UvrD, and pCRA helicases. The result of Tth-UvrD helicase is missing a recognition site compared with the others, and Tte-Uvr D helicase can obtain more balanced and unbiased results.

[0112] Example 7 HPA reaction using phi 29 DNA polymerase

[0113] The HPA reaction was performed using phi 29 DNA polymerase instead of BSt polymerase.

[0114] The low-copy number 9948 genome was amplified and recovered using a recovery kit. The recovered product was used as template for amplification verification using the Xinhai Biotech Human Genome STR Locus 25A Forensic Kit and capillary electrophoresis. The HPA reaction system consisted of: 2 μL 10× HPA reaction buffer, 0.4 μL 25 mM dNTPs, 1 μL 100 mM ATP, 0.2 μL 1 M DTT, 0.5 μL 0.1 mg / mL Tth-primpol, 1 μL phi29 DNA polymerase, 1 μL Tte UvrD helicase (0.2 mg / mL), 0.7 μL 5 mg / mL E. coli SSB, 1 μL DMSO, and 0.2 μL 4 M ammonium sulfate solution. 7 μL of 5 pg / μL template was added to the reaction system, and the reaction was incubated at 42°C for 60 min. After the reaction, the gel recovery kit was used to recover the product, and the recovered product was used as a template to amplify the human genome ST R site 25A forensic kit and analyzed by capillary electrophoresis. Figure 9 It can be seen that when amplifying a 5pg / μL genome, using phi29 to perform HPA treatment on the genome can increase the amount of the genome and significantly increase the peak height detected by the human genome STR site 25A forensic kit.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for isothermal amplification of nucleic acid molecules, characterized in that The steps include: S1: Mix the nucleic acid to be amplified with helicase to obtain single-stranded nucleic acid; S2: the single-stranded nucleic acid is mixed with a single-stranded binding protein and a primer enzyme to obtain an oligonucleotide; S3: In the presence of dNTPs, the oligonucleotide is mixed with DNA polymerase to obtain an extension product; S4: The extension product repeats S1 to S3 above once or multiple times to obtain the sequence of the nucleic acid to be amplified.

2. The method according to claim 1, wherein The method does not include the step of adding primers and / or primer sets.

3. The method according to claim 1 or 2, wherein: The mixing described in S1 also includes a step of mixing with ATP.

4. The method according to any one of claims 1 to 3, wherein The helicase includes any one of the UvrD helicase derived from Thermusthermophilus, the UvrD helicase derived from Thermoanaerobacter te ngcongensis, and the PcrA helicase derived from Geobacillus stearothermophilus.

5. The method according to any one of claims 1 to 4, characterized in that The primer enzyme includes: DnaG type primer enzyme or AEP type primer enzyme.

6. The method according to any one of claims 1 to 5, characterized in that The single-chain binding protein is derived from Escherichia coli or Thermus thermophilus.

7. The method according to any one of claims 1 to 6, wherein: The DNA polymerase includes: phi 29 DNA polymerase or Bst3.0 DNA polymerase.

8. The method according to any one of claims 1 to 7, wherein: The isothermal amplification time is 40 to 60 minutes, and the temperature is 42 to 65°C.

9. An enzyme composition, characterized in that include: Helicases, primases, DNA polymerases, and single-strand binding proteins.

10. A reaction reagent, characterized in that include: The enzyme composition of claim 9, dNTPs, ATP, DTT, and a buffer; The reaction reagents do not include: primers and / or primer sets.

11. The product, characterized in that include: The enzyme composition according to claim 9 and / or the reaction reagent according to claim 10 and acceptable auxiliary agents.

12. Use of the enzyme composition according to claim 9, the reaction reagent according to claim 10 and / or the product according to claim 11 in isothermal amplification.

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