Mutant Taq DNA polymerase and its application

Abstract

The present invention belongs to the field of molecular biology technology, and specifically discloses a mutant Taq DNA polymerase and its application. The present invention improves the amplification performance of Taq DNA polymerase as a whole, including high GC, long fragment and rapid amplification ability, as well as high salt tolerance, low copy amplification and A addition ability, by introducing mutation combination 1 and / or 2 into the structural domain of wild-type Taq DNA polymerase. Among them, the amplification performance of mutants M4, M9, M10, M11, M14, and M15 is better than that of other mutants, and each has advantages over the reported mutant CK in high salt tolerance, A addition efficiency, rapid amplification, and low copy amplification. The mutant Taq DNA polymerase of the present invention can be used to prepare conventional PCR and multiplex PCR detection reagents, sequencing library construction reagents, genotype identification reagents, site-directed mutagenesis reagents, etc., and has a wide range of applications in the fields of molecular diagnosis and precision medicine testing.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a mutant Taq DNA polymerase and an application thereof. Background Art

[0002] Taq DNA polymerase is a heat-resistant DNA polymerase belonging to the DNA polymerase I family. The gene is 2496bp long, encoding 832 amino acids and has a molecular weight of 94kD. Studies have shown that Taq DNA polymerase is divided into three domains, among which the first domain is composed of amino acids 1-291 at the N-terminus, expressing 5'→3' exonuclease activity. At the same time, the binding site for divalent metal ions is also located in this region; the second domain is composed of amino acids 424-832 at the C-terminus, expressing 5'→3' polymerase activity. This region can also be figuratively compared to a person's right hand, including the thumb domain, finger domain and palm domain; the third domain is located in the middle part of the enzyme protein peptide chain, namely amino acids 292-423. This region has structural homology with E. coli DNA polymerase I, but lacks sequence homology, so Taq DNA polymerase does not have the 3'→5' exonuclease activity of E. coli DNA polymerase I.

[0003] Studies have reported that Taq DNA polymerase has good polymerization activity. Within a certain range, the higher the temperature, the higher the enzyme synthesis rate. However, too high (above 90°C) or too low (22°C) temperatures will affect the activity of the enzyme. Under 70-75°C conditions, Taq DNA polymerase has the best polymerase activity, while the enzyme activity only lasts for 130 minutes at 92.5°C and 40 minutes at 95°C. In addition, Taq DNA polymerase also has an activity similar to terminal deoxyribonucleic acid transferase (TdT), which can add a base to the 3' end of the newly synthesized double strand. Although all four bases can be added to the 3' end, Taq DNA polymerase has a much higher polymerization ability for dATP than the other three bases, so under standard PCR conditions, the non-template-dependent polymerization base at the 3' end of the product is almost always A, and is therefore also suitable for TA cloning.

[0004] At present, the modification and transformation of Taq DNA polymerase mainly includes: 1. Improving the processability of the enzyme, for example, increasing the binding ability of the enzyme to the template by site-directed mutagenesis (S543N), reducing the sensitivity of the enzyme to the template structure, thereby improving the amplification ability of Taq DNA polymerase for difficult templates (such as high GC content) and long fragments (greater than 2kb), or fusing Taq DNA polymerase with the non-sequence-specific dsDNA binding protein Sso7d by genetic or protein engineering to enhance the binding of the enzyme to the template, thereby improving the processivity of the enzyme; 2. Improving the fidelity of the enzyme, for example, by deletion mutation (deletion of amino acids 1-289 at the N-terminus) or site-directed mutagenesis (G46D), or inserting T3 in the thumb domain of the polymerase 1. To improve the fidelity of the enzyme by modifying the thioredoxin domain of the DNA polymerase, while improving the enzyme's heat resistance and ability to bind to the template; 2. To improve the enzyme's tolerance to inhibitors, for example, by increasing the extension rate of Taq DNA polymerase through combined mutations (such as Taq-E7S mutants: K53N, K56Q, E57D and E507K), thereby improving tolerance to plant-derived inhibitors, blood inhibitors, etc., making it suitable for direct sample amplification; 3. To improve the amplification specificity of the enzyme, by increasing the optimal reaction temperature of the enzyme through chemical modification, nucleic acid aptamers or antibody-specific binding, weakening the enzyme's amplification ability at low temperatures, thereby improving amplification specificity, etc.

[0005] U.S. Patent No. 9315787B2 (Kapa Biosystems) discloses a mutant Taq DNA polymerase, which improves the tolerance of the enzyme to plant-derived inhibitors (such as salt, polysaccharides, polyphenols, secondary metabolites, etc.) by combining mutations (A61T, K346E, S357C, I707M and F749I), and the amplification ability is significantly better than that of the Taq-E7S mutant. In addition, Chinese Patent No. CN106754812B (Novozyme) discloses a Taq DNA polymerase for NGS library construction, which improves the enzyme's A addition ability and library construction efficiency by combining mutations (E315K, E388V, E507K, D578G, A608V and M747R), is suitable for templates with lower starting amounts, and increases the integrity and coverage of library construction. However, the modification and transformation of Taq DNA polymerase in the above patents is limited to improving the enzyme activity in a certain aspect, and cannot improve the amplification performance of the enzyme as a whole. Therefore, it is necessary to develop a new type of Taq DNA polymerase that can increase the enzyme's extension rate while improving the enzyme's inhibitor tolerance and A-addition ability, thereby improving the enzyme's ability to amplify difficult templates. Summary of the invention

[0006] The main technical problem solved by the present invention is to provide a mutant Taq DNA polymerase, which increases the enzyme extension rate while improving the enzyme's inhibitor tolerance and A addition ability, improves the enzyme's amplification ability for difficult templates, and improves the enzyme's amplification performance as a whole.

[0007] Secondly, the present invention provides a gene encoding a mutant Taq DNA polymerase.

[0008] Thirdly, the present invention provides a biological material comprising a mutant Taq DNA polymerase or a gene encoding the mutant Taq DNA polymerase.

[0009] Finally, the present invention provides a mutant Taq DNA polymerase or its encoding gene, biological material for use in preparing conventional PCR detection reagents, multiplex PCR detection reagents, sequencing library construction reagents, genotype identification reagents, site-directed mutagenesis reagents, etc.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A mutant Taq DNA polymerase, compared with the amino acid sequence of a wild-type Taq DNA polymerase (as shown in SEQ ID NO: 1), comprises mutation combination 1 or mutation combination 2:

[0012] The mutation combination 1 comprises the following mutation sites:

[0013] Mutation site 4: E400K, i.e., glutamic acid at position 400 mutated to lysine;

[0014] Mutation site 5: D452N, i.e., aspartic acid at position 452 mutated to asparagine;

[0015] Mutation site 6: E507K, i.e., glutamic acid at position 507 mutated to lysine;

[0016] The mutation combination 2 comprises the following mutation sites:

[0017] Mutation site 1: K53N, i.e., lysine at position 53 mutates to asparagine;

[0018] Mutation site 2: K56Q, i.e., lysine at position 56 mutates to glutamine;

[0019] Mutation site 3: E57D, i.e., glutamic acid at position 57 mutated to aspartic acid;

[0020] Mutation site 6: E507K, i.e., glutamic acid at position 507 mutated to lysine;

[0021] Mutation site 7: L552R, i.e., the leucine at position 552 mutates to arginine.

[0022] As a preferred embodiment of the present invention, the mutant Taq DNA polymerase has a sequence homology of more than 90% with the wild-type Taq DNA polymerase, and more preferably has a sequence homology of more than 95%, more than 96%, more than 97%, more than 98% or more than 99%.

[0023] As a preferred embodiment of the present invention, the mutant Taq DNA polymerase comprises mutation combination 1 compared with the amino acid sequence of the wild-type Taq DNA polymerase:

[0024] The mutation combination 1 comprises the following mutation sites:

[0025] Mutation site 4: E400K;

[0026] Mutation site 5: D452N;

[0027] Mutation site 6: E507K;

[0028] Mutation site 7: L552R; and / or,

[0029] Mutation site 8: I707L, i.e., the isoleucine at position 707 mutated to leucine.

[0030] As a preferred embodiment of the present invention, the mutant Taq DNA polymerase includes a non-substitution modification, preferably a reversibly inactivated hot-start modification, including but not limited to chemical modification, nucleic acid aptamer modification, affinity antibody modification, etc. The modified mutant Taq DNA polymerase loses (or mostly loses) polymerase activity at low temperatures, the hot-start modification is destroyed at high temperatures, and the polymerase activity is reactivated, thereby being suitable for hot-start PCR.

[0031] Specifically, the chemically modified mutant Taq DNA polymerase can be prepared according to patent CN1282741C (Roche), that is, the mutant Taq DNA polymerase is reacted with a dicarboxylic anhydride having a structure of formula (I) or formula (II) under alkaline pH and reaction conditions below 25°C.

[0032] The dicarboxylic anhydride having the structure of formula (I) or formula (II) is shown below:

[0033]

[0034] In formula I, R1 and R2 are each independently selected from hydrogen or an organic group, and they may be connected;

[0035] In formula II, R1 and R2 are each independently selected from an organic group, they may be linked, and hydrogen is cis.

[0036] The organic group can be directly connected to the ring through a carbon-carbon bond, a carbon-heteroatom bond (such as a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond). The organic groups can also be connected to each other to form a ring structure, for example, 3,4,5,6-tetrahydrophthalic anhydride.

[0037] Specifically, the mutant Taq DNA polymerase modified by the nucleic acid aptamer can be prepared with reference to patents CN101768616B (Roche) and US9109226B2 (NEB). Nucleic acid aptamers are generally obtained by screening using the SELEX technology. Their inhibitory properties on DNA polymerase do not depend on the exact nucleotide sequence, but on the conformation or shape of the secondary structure formed by the sequence, as well as the melting temperature of the structure.

[0038] Specifically, the mutant Taq DNA polymerase modified with the affinity antibody can be prepared with reference to patents EP726324B1 (Johnson & Johnson) and CN107119029A (Hailite), and the affinity antibody is obtained by immune screening.

[0039] As a preferred embodiment of the present invention, the mutant Taq DNA polymerase is fused with Sso7d, PCNA, or with a protein tag (such as a His tag, a GST tag) to obtain a fusion protein with improved enzyme activity.

[0040] A gene encoding a mutant Taq DNA polymerase.

[0041] As a preferred embodiment of the present invention, without changing the amino acid sequence of the encoded mutant Taq DNA polymerase, the codon preference of the host cell can be optimized.

[0042] A biological material comprising a mutant Taq DNA polymerase or a gene encoding the mutant Taq DNA polymerase.

[0043] As a preferred embodiment of the present invention, the biological material includes but is not limited to a recombinant expression vector (such as a plasmid vector), a gene expression cassette, a recombinant bacterium, and the like.

[0044] As a preferred embodiment of the present invention, the method for preparing the recombinant expression vector comprises: introducing a gene encoding a mutant Taq DNA polymerase into the expression vector to obtain the recombinant expression vector.

[0045] As a preferred embodiment of the present invention, the method for preparing the recombinant bacteria comprises: transforming a recombinant expression vector containing a gene encoding a mutant TaqDNA polymerase into a host bacterium (such as Escherichia coli) to obtain the recombinant bacteria.

[0046] Specifically, the preparation method of the recombinant bacteria includes the following steps: transforming the recombinant expression vector into Escherichia coli and spreading it on an agar medium containing drugs such as ampicillin, inoculating the colonies into LB medium or 2×YT medium after the colonies are formed, and culturing them at 37°C for 12-20 hours, then crushing the bacteria (physical crushing methods such as ultrasonic crushing, French press crushing, glass bead crushing, or lysozyme lysis can be used), and extracting to obtain a crude enzyme solution; heat-treating the crude enzyme solution (for example, treating it at 80°C for 30 minutes), then removing nucleic acids with PEI, and then removing some impurities with ammonium sulfate precipitation, recovering the DNA polymerase fraction, and then desalting it with Sephadex G-25 gel filtration and other methods, separating and purifying it with a heparin agarose column to obtain a purified enzyme sample.

[0047] A mutant Taq DNA polymerase or its encoding gene and biological material.

[0048] As a preferred embodiment of the present invention, the use includes one or more of the following:

[0049] (1) Application in the preparation of conventional PCR detection reagents or kits;

[0050] (2) Application in the preparation of multiplex PCR detection reagents or kits;

[0051] (3) Application in the preparation of sequencing library construction reagents or kits;

[0052] (4) Application in the preparation of genotype identification reagents or kits;

[0053] (5) Application in the preparation of site-directed mutagenesis reagents or kits.

[0054] Specifically, the conventional PCR detection reagent or kit, the multiplex PCR detection reagent or kit also includes deoxyribonucleoside triphosphates: dATP, dGTP, dTTP and dCTP, with concentrations of 0.1-0.6 mM respectively. The detection reagent or kit may also include one or more of the following components:

[0055] (1)Mg 2+ Activators, such as MgCl2, MgSO4;

[0056] (2) Buffer substances, such as Tris-HCl, Tris-H2SO4, etc.;

[0057] (3) Cosolvents, such as formamide, dimethyl sulfoxide (DMSO), ethylene glycol, 1,2-propylene glycol, polyethylene glycol (PEG), etc.

[0058] (4) Additives, such as NaCl, KCl, EDTA, betaine, tetramethylammonium chloride TMAC, ammonium chloride, ammonium sulfate, ammonium acetate, glycerol, bovine serum albumin BSA, gelatin, dithiothreitol DTT, cationic or non-ionic detergents (such as Tween-20, Triton X-100, NP-40), etc.

[0059] Specifically, the mutant Taq DNA polymerase in the sequencing library construction reagent or kit can be used for the end-repair and A-addition reaction after fragmentation, or for multiple PCR amplification of multiple target genes. When used for the end-repair and A-addition reaction, the concentration of dATP in the reagent or kit can be appropriately increased to facilitate the A-addition reaction. The reagent or kit can also include a variety of enzymes that can form blunt ends, 5' end phosphorylation or 3' end dephosphorylation of DNA fragments. For example, the reagent or kit includes an enzyme complex composed of one or more of the following enzymes: T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, T4 polynucleotide kinase. The reagent or kit also includes one or more of the following components: Tris-HCl, MgCl2, a monovalent metal salt selected from NaCl, KCl, LiCl, ATP, Tween-20, Triton X-100, NP-40, glycerol, DTT, EDTA, etc. When used for multiple PCR amplification, the reagent or kit can also include components that can improve amplification uniformity and specificity, including but not limited to betaine, DMSO, TMAC, BSA, KCl, glycerol, etc.

[0060] A conventional PCR detection kit, a multiplex PCR detection kit or a sequencing library construction kit comprises: at least one container containing a mutant Taq DNA polymerase.

[0061] As a preferred embodiment of the present invention, the mutant Taq DNA polymerase and the reaction buffer together form a reaction premix, such as a 2×Mix form. When used, the reaction premix only needs to be mixed with primers and templates, and water is added to prepare a reaction system (such as diluted to 1×).

[0062] As a preferred embodiment of the present invention, the kit further comprises another container, wherein the another container comprises a reaction buffer, that is, the mutant Taq DNA polymerase and the reaction buffer are provided separately. Different from the above-mentioned product form of providing a reaction premix, the kit needs to take the mutant Taq DNA polymerase and the reaction buffer separately when in use, and then mix them with primers and templates, and then add water to prepare a reaction system.

[0063] Specifically, the reaction buffer contains all components required for the reaction, including but not limited to dNTPs, Mg 2+One or more of activators, buffer substances, co-solvents, additives, etc. (same types as above).

[0064] Beneficial effects of the present invention:

[0065] The present invention provides a mutant Taq DNA polymerase or a gene encoding the same, and by introducing a mutation combination 1 and / or a mutation combination 2 into the structural domain of a wild-type Taq DNA polymerase, the amplification performance of the Taq DNA polymerase is improved as a whole, including high GC, long fragment and rapid amplification capabilities, as well as high salt tolerance, low copy amplification and A addition capabilities, etc. Among them, the amplification performance of mutants M4, M9, M10, M11, M14 and M15 is better than that of other mutants, and each has advantages over the reported mutant CK in terms of high salt tolerance, A addition efficiency, rapid amplification and low copy amplification.

[0066] The present invention also provides a biological material comprising a mutant Taq DNA polymerase or a gene encoding it, which can be used to prepare conventional PCR detection reagents, multiplex PCR detection reagents, sequencing library construction reagents, genotype identification reagents, site-directed mutagenesis reagents, etc., and has a wide range of applications in the fields of molecular diagnosis and precision medicine testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the amplification result of the mutant in the experimental example for the template with 28% GC content.

[0068] Figure 2 This is the amplification result of the mutant in the experimental example for a template with 54% GC content.

[0069] Figure 3 It is the amplification result of the mutant on the template with 78% GC content in the experimental example;

[0070] In the figure, A: no DMSO was added, B: 5% DMSO was added.

[0071] Figure 4 This is the amplification result of the long fragment by the mutant in the experimental example.

[0072] Figure 5 This is the result of rapid amplification of mutants in the experimental example;

[0073] In the figure, A: human genomic DNA as template, B: λDNA as template.

[0074] Figure 6 It is the amplification result of the mutant in the experimental example in the high-salt reaction buffer;

[0075] In the figure, A: buffer A + 150mM KCl, B: buffer A + 150mM NaCl.

[0076] Figure 7 It is the amplification result of the mutant in the experimental example with different concentrations of template;

[0077] In the figure, A: 100 ng / μL template, B: 10 ng / μL template, C: 100 pg / μL template, D: 10 pg / μL template.

[0078] Figure 8 These are the experimental results of the A addition efficiency of the mutants in the experimental example.

[0079] In order to more clearly illustrate the technical solution to be protected by the present invention, the above drawings obtained in the experimental examples are briefly introduced. It should be understood that the above drawings only show the technical solutions and technical effects of certain experimental examples of the present invention, and should not be regarded as any limitation on the scope of protection of the claims. For ordinary technicians in this field, other relevant drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0080] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solution of the present invention and should not be regarded as any limitation on the scope of protection of the claims. Based on the following embodiments and experimental examples, other technical solutions obtained by ordinary technicians in the field without paying creative work, such as technical solutions obtained after modification, denaturation or simple replacement, should all belong to the scope of protection of the present invention. In the absence of conflict, the technical features in different embodiments can be combined and replaced with each other.

[0081] The conventional methods of molecular biology and nucleic acid chemistry used in the following embodiments and experimental examples are all prior arts and are described in detail in the literature, see "A Guide to Molecular Cloning", Sambrook et al., 1989; "Oligonucleotide Synthesis", MJ Gait et al., 1984; "Nudeic Acid Hybridization", BD Hames and SJ Higgins, 1984; "Basic Methods in Molecular Biology", Elsevier; "Current Protocols in Molecular Biology", John Wiley and Sons; "Methods in Enzymology", American Academic Press; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art, easily available to the public or available through commercial channels unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS is phosphate buffered saline.

[0082] The term "gene" refers to a DNA sequence containing the control and coding sequences necessary for the production of a recoverable biologically active polypeptide or precursor.

[0083] The term "wild type" refers to a gene or gene product isolated from a natural source. The term also refers to a recombinant form of a natural protein prepared by molecular biology techniques, whose amino acid sequence is identical to that of the natural protein.

[0084] The term "mutant" refers to a gene whose nucleotide sequence has been altered or a gene product whose amino acid sequence has been altered, resulting in a gene product having functional properties different from those of the native or wild-type gene or gene product.

[0085] The term "mutation" refers to a single amino acid substitution, and point mutations are preferably introduced into the amino acid sequence by changing the appropriate codons in the encoding DNA. The amino acid positions are numbered according to the full-length sequence of the protein, and the regions containing the mutations are from the full-length sequence. Nucleotides and point mutations in DNA sequences are represented similarly.

[0086] Example

[0087] This embodiment provides a mutant Taq DNA polymerase (amino acid sequence as shown in SEQ ID NO: 3, nucleotide sequence as shown in SEQ ID NO: 4), which comprises mutation combination 1 compared with the amino acid sequence of wild-type Taq DNA polymerase (as shown in SEQ ID NO: 1):

[0088] Mutation site 4: E400K, i.e., glutamic acid at position 400 mutated to lysine;

[0089] Mutation site 5: D452N, i.e., aspartic acid at position 452 mutated to asparagine;

[0090] Mutation site 6: E507K, i.e., glutamic acid at position 507 mutates to lysine.

[0091] This embodiment also provides a method for preparing the mutant Taq DNA polymerase, comprising the following steps:

[0092] 1. Construction of recombinant strains

[0093] (1) Acquisition of target genes

[0094] Using the plasmid containing the wild-type Taq DNA polymerase gene (as shown in SEQ ID NO: 2) synthesized by Bio-Tech as a template, PCR amplification was performed using primer pairs A12-F1 / A12-R1, A12-F2 / A5-R, and A5-F / A12-R2 (as shown in Table 4), respectively, and the amplification reaction system is shown in Table 1. The amplification reaction program was: pre-reaction at 95°C for 5 min, followed by 95°C, 15 s, 65°C, 15 s, 72°C, 1 kb / min, 25 cycles, to obtain the target gene fragment with the mutation site.

[0095] Table 1 Amplification reaction system

[0096] Components Addition volume μL Template DNA 0.5 10 μM Primer F 1 10 μM Primer R 1 PrimeSTAR 0.5 dNTPs(10mM) 1 5×PS buffer 10 <![CDATA[ddH2O]]> 36

[0097] (2) Seamless cloning

[0098] The amplified target gene with mutation site and expression vector pET-30a were mixed at a molar ratio of 1:1, and then 2×ClonExpress Mix (Novozyme) was added and reacted at 50°C for 10 min.

[0099] (3) Conversion

[0100] The ligation product was transformed into the expression host BL21 (DE3), plated for culture, and then positive clones were screened by colony PCR, and recombinant strains containing the corresponding mutation sites were obtained by sequencing. The PCR screening system is shown in Table 2, which was divided into 14 μL / tube, and 1 μL of bacterial solution was added for detection. The program was: 95°C pre-reaction for 5 min, followed by 95°C, 30 s, 65°C, 30 s, 72°C, 2 min 50 s, 28 cycles, and agarose gel electrophoresis was performed after the reaction to screen positive clones.

[0101] Table 2 PCR screening system

[0102]

[0103]

[0104] 2. Expression and purification of recombinant proteins

[0105] The recombinant strain with correct sequencing was inoculated and fermented for preparing bacteria. It was inoculated into LB medium at an inoculum of 1%, cultured at 37°C with shaking until OD600 was 0.6-0.8, 1mM IPTG was added for induction, and the bacteria were collected after continuing to culture for 4 hours. The bacteria were resuspended in a buffer solution (composed of: 50mM Tris-HCl, pH=7.5, 0.3M NaCl, 1mM EDTA, 1mM DTT) at a ratio of 1:20, the cells were broken by ultrasonic treatment, the supernatant was collected by centrifugation, the supernatant was heat-treated at 65°C for 30 minutes, and the insoluble substances were removed by centrifugation to obtain a crude sample from which most of the impurities were removed, which could be used for screening and detection of mutants.

[0106] In other embodiments of the present invention, mutants shown in Table 3 were constructed according to the same method as in Example 1, and the primers corresponding to each mutation site were shown in Table 4.

[0107] Table 3 Constructed Taq DNA polymerase mutants

[0108] Mutants Mutation site M1 E315K M2 E400K M3 D452N M4 E507K、L552R M5 H676R M6 I707L M7 D732G M8 F749V M9 K53N, K56Q, E57D, E507K, L552R M10 E400K, D452N, E507K M11 E400K, D452N, E507K, I707L A7 E742K CK K53N, K56Q, E57D, E507K M14 E400K, D452N, E507K, L552R, I707L M15 E400K, D452N, E507K, L552R

[0109] Table 4 Primers corresponding to each mutation site and primers used in other experimental examples

[0110]

[0111]

[0112]

[0113] Experimental example

[0114] 1. Evaluation of amplification ability of templates with different GC contents

[0115] Using human genomic DNA as a template, 28% GC, 54% GC, and 78% GC amplification primers (as shown in Table 4) were designed and synthesized for PCR amplification. The amplification reaction system is shown in Table 5, and the amplification reaction procedure is shown in Table 6. After the amplification is completed, agarose gel electrophoresis is performed to evaluate the amplification ability of the mutants for templates with different GC contents based on the electrophoresis results. Among them, a group of 78% GC amplification reaction systems was set up to add 5% DMSO to enhance the amplification effect. The results are shown in Table 6. Figure 1-3 , as shown in Table 9.

[0116] Table 5 Amplification reaction system

[0117]

[0118]

[0119] In the table, the composition of 5×buffer A is: 100mM Tris-HCl, 250mM KCl, 15mM MgCl2, 25℃, pH 8.3.

[0120] Table 6 Amplification reaction program

[0121]

[0122] 2. Evaluation of long fragment amplification ability

[0123] Using λDNA as a template, 5kb, 8kb, and 10kb amplification primers (as shown in Table 4) were designed and synthesized for PCR amplification. The amplification reaction system was shown in Table 5, and the amplification reaction procedure was shown in Table 6. The amplification was extended at 72°C for 5 min. After the amplification was completed, agarose gel electrophoresis was performed to evaluate the amplification ability of the mutant for long fragments. The results are shown in Figure 4 , as shown in Table 9.

[0124] 3. Rapid expansion capability evaluation

[0125] Human genomic DNA or λDNA was used as a template, and PCR amplification was performed using a 54% GC amplification primer and a designed and synthesized λDNA 1kb amplification primer (as shown in Table 4), respectively. The amplification reaction system was shown in Table 5, and the amplification reaction program was shown in Table 6. The extension was performed at 72°C for 4s or 2s. After the amplification was completed, agarose gel electrophoresis was performed to evaluate the rapid amplification ability of the mutant. The results are shown in Table 6. Figure 5 , as shown in Table 9.

[0126] 4. Inhibitor tolerance evaluation

[0127] λDNA was used as a template and PCR amplification was performed using λDNA1kb amplification primers. The amplification reaction system was shown in Table 5. Buffer A+150mM KCl or buffer A+150mM NaCl was used. The amplification reaction program was shown in Table 6. The extension was performed at 72°C for 30s. After the amplification was completed, agarose gel electrophoresis was performed to evaluate the inhibitor tolerance of the mutant. The results are shown in Figure 6 , as shown in Table 9.

[0128] 5. Evaluation of amplification ability of templates at different concentrations

[0129] Human genomic DNA was used as a template, and the template concentrations were set to 100 ng / μL, 10 ng / μL, 100 pg / μL, and 10 pg / μL, respectively. PCR amplification was performed using 54% GC amplification primers (product length was about 1 kb). The amplification reaction system was shown in Table 5, and the amplification reaction procedure was shown in Table 6. After the amplification was completed, agarose gel electrophoresis was performed to detect the mutants. The amplification ability of the mutants for templates of different concentrations was evaluated based on the electrophoresis results. The results are shown in Table 5. Figure 7 , as shown in Table 9.

[0130] 6. Add A ability evaluation

[0131] The A addition efficiency of each mutant was detected by adding A to the 3' end of the blunt-ended hairpin structure ON0. First, the ON0 hairpin structure was prepared by annealing, and the prepared annealing reaction system (as shown in Table 7) was annealed according to the set program (program: 95°C, 5min, 94°C to 37°C, and 1°C was reduced every 30s) to obtain the reaction substrate.

[0132] Table 7 Annealing reaction system

[0133] Components Add 100 μM ON0 50μL 10×T4 PNK buffer(NEB) 10μL <![CDATA[ddH2O]]> Make up to 100 μL

[0134] Using the reaction substrate as a template, a reaction system for determining the efficiency of A addition was prepared (as shown in Table 8), and the A addition reaction was carried out according to the set reaction program (program: 68° C. reaction for 30 min). After the reaction was completed, an equal volume of 25 mM EDTA was added to terminate the reaction.

[0135] Table 8 Reaction system for determining the efficiency of adding A

[0136] Components Add Wild-type Taq or mutant 2.5μL 5×buffer A 5μL Annealing ON0 (50μM) 2μL dATP(2.5mM) 4μL <![CDATA[ddH2O]]> Make up to 50 μL

[0137] After the addition of A reaction was completed, HPLC detection was performed (C18 column, 25% acetonitrile, flow rate of 0.5 mL / min). HPLC detection can separate the substrate and product with a difference of one base. The retention time of the substrate peak is about 5.9 min, and the retention time of the product peak is about 5.8 min. Then, the addition efficiency of different mutants was calculated according to the peak area ratio. The results are as follows: Figure 8 , as shown in Table 9.

[0138] Table 9 Evaluation results of various properties of mutants

[0139]

[0140]

[0141] It can be seen from the above experimental results that the present invention improves the amplification performance of Taq DNA polymerase as a whole, including high GC, long fragment and rapid amplification ability, as well as high salt tolerance, low copy amplification, A addition ability, etc., by introducing mutation combination 1 and / or mutation combination 2 into the structural domain of wild-type Taq DNA polymerase. Among them, the amplification performance of mutants M4, M9, M10, M11, M14, and M15 is better than that of other mutants (preliminary experimental results show that the amplification performance of mutant M4 is significantly better than that of Taq DNA polymerase with single point mutations of E507K or L552R), and has advantages over the reported mutant CK in high salt tolerance, A addition efficiency, rapid amplification, and low copy amplification.

[0142] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments and experimental examples, for ordinary technicians in this field, modifications, substitutions or improvements made without departing from the spirit of the present invention should all fall within the scope of protection of the present invention.

Claims

1. A mutant Taq DNA polymerase, Features: Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase has only any one of the following mutation combinations: Mutation combination A: E400K, D452N, and E507K; Mutation combination B: E400K, D452N, E507K, and L552R; Mutation combination C: E400K, D452N, E507K, and I707L; Mutation combination D: E400K, D452N, E507K, L552R, and I707L; The amino acid sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO:

1.

2. A gene encoding the mutant Taq DNA polymerase as claimed in claim 1.

3. A biological material comprising the mutant Taq DNA polymerase according to claim 1, or the encoding gene according to claim 2, Features: The biological material is a recombinant expression vector, a gene expression cassette or a recombinant bacterium.

4. A use of the mutant Taq DNA polymerase according to claim 1, or the encoding gene according to claim 2, or the biomaterial according to claim 3, Features: The uses include one or more of the following: (1) Application in the preparation of conventional PCR detection reagents; (2) Application in the preparation of multiplex PCR detection reagents; (3) Application in the preparation of sequencing library construction reagents; (4) Application in the preparation of genotype identification reagents; (5) Application in the preparation of site-directed mutagenesis reagents.

5. A conventional PCR detection kit, a multiplex PCR detection kit or a sequencing library construction kit, It is characterized in that The kit comprises: at least one container, wherein the container contains the mutant Taq DNA polymerase according to claim 1.

6. The kit according to claim 5, Features: The mutant Taq DNA polymerase and the reaction buffer in the kit together form a reaction premix; Alternatively, the kit further comprises another container comprising a reaction buffer.

7. The kit according to claim 5, Features: The conventional PCR detection kit or the multiplex PCR detection kit also includes deoxyribonucleoside triphosphates: dATP, dGTP, dTTP and dCTP, with concentrations of 0.1-0.6 mM respectively; And / or, the conventional PCR detection kit or the multiplex PCR detection kit further comprises one or more of the following components: (1) Mg 2+ Activators, including but not limited to MgCl 2 MgSO 4 ; (2) Buffer substances, including but not limited to Tris-HCl, Tris-H 2 SO 4 ; (3) Solvents, including but not limited to formamide, dimethyl sulfoxide, ethylene glycol, 1,2-propylene glycol, and polyethylene glycol; (4) Additives, including but not limited to NaCl, KCl, EDTA, betaine, tetramethylammonium chloride, ammonium chloride, ammonium sulfate, ammonium acetate, glycerol, bovine serum albumin, gelatin, dithiothreitol, cationic or non-ionic detergents.

8. The kit according to claim 5, Features: The mutant Taq DNA polymerase in the sequencing library construction kit is used for the end-repair and A addition reaction after fragmentation, or for multiplex PCR amplification of multiple target genes; When used for the final addition A reaction, the concentration of dATP in the kit is higher than that of dGTP, dTTP and dCTP; When used for multiplex PCR amplification of multiple target genes, the kit further comprises one or more of the following components: betaine, DMSO, TMAC, BSA, KCl, and glycerol.

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