DNA polymerase thermal stability mutant based on rational design and construction method thereof
Through rational design, single-point mutants of Phi29 DNA polymerase, namely H149D, T203D, R306I, N313P, and K422E, were constructed, solving the problem of insufficient thermal stability and enabling efficient application under high-temperature conditions.
Patent Information
- Application Number
- CN202511569823.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-30
- Publication Date
- 2025-12-26
AI Technical Summary
The existing Phi29 DNA polymerase has insufficient thermostability, which limits its application in high-temperature reaction systems.
Using a rational design approach, based on multiple sequence alignment, phylogenetic analysis, and three-dimensional structural information, five single-point mutants, including H149D, T203D, R306I, N313P, and K422E, were identified and constructed to improve their thermal stability.
The thermostability of Phi29 DNA polymerase has been significantly improved, making it perform better in the range of 40℃-70℃, and suitable for high-temperature amplification and processing of templates with high GC content.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a DNA polymerase thermal stability mutant based on rational design and a construction method thereof. BACKGROUND
[0002] Phi29 DNA polymerase is derived from Bacillus subtilis bacteriophage, and its characteristic functions mainly include three aspects: first, high-efficiency DNA template-dependent catalytic activity, which can drive dNTP polymerization to synthesize complementary DNA strands; second, strong strand displacement ability, which can realize the dissociation of double-stranded DNA without the assistance of helicase; and third, excellent high fidelity, which has extremely low replication error rate, thereby ensuring the sequence accuracy of the amplification product. Based on these characteristics, the enzyme has been widely used in rolling circle amplification and whole genome amplification, and has shown significant advantages in dealing with nucleic acid templates with complex secondary structures. However, the thermal stability of wild-type Phi29 DNA polymerase is its key shortcoming (it is easily inactivated above 60℃), which constitutes a bottleneck for its application in high-temperature reaction systems (such as rapid high-temperature amplification or high-GC content template processing).
[0003] At the level of structural biology, there is often significant degeneracy between the primary sequence of a protein and its spatial conformation, which makes a single sequence can adopt multiple stable and energy similar folding states. This principle lays the foundation for rational design-based protein engineering, that is, through computer simulation and site-directed mutagenesis technology, functional properties such as thermal stability, catalytic efficiency and environmental tolerance can be targetedly optimized. Among them, "consensus design" is a rational strategy based on evolutionary information, which assumes that the amino acid residues with the highest frequency in a conserved site in a homologous protein family are most conducive to maintaining the stability of the protein structure. In this study, guided by this theoretical framework, multi-sequence alignment, phylogenetic analysis and three-dimensional structure information were integrated to conduct a comprehensive bioinformatics scan of the Phi29 DNA polymerase family. Through this rational design process, the present application successfully identified and constructed five single-point mutants, and experiments confirmed that the thermal stability of all of them was significantly improved compared with the wild type. SUMMARY
[0004] The technical problem to be solved by the present application is to improve the thermal stability of the existing Phi29 DNA polymerase, thereby providing a DNA polymerase thermal stability mutant based on rational design and a construction method thereof.
[0005] To solve the above technical problems, the technical scheme adopted by the present application is as follows: the present application provides a DNA polymerase thermal stability mutant based on rational design, which is as follows (a1) or (a2): (a1) a derivative protein in which one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2 are substituted, deleted or added and which has the same function as the amino acid sequence shown in SEQ ID NO. 2; (a2) a derivative protein in which one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2 are substituted, deleted or added and which has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2.
[0006] Preferably, the DNA polymerase thermal stability mutant comprises a single point mutant at any one of the single point mutation sites of H149D, T203D, R306I, N313P, K422E in the amino acid sequence shown in SEQ ID NO. 2.
[0007] Preferably, the DNA polymerase thermal stability mutant obtained by the mutation sites of H149D, T203D, R306I, N313P, K422E has the amino acid sequences of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, respectively.
[0008] The present application provides a gene encoding the rationally designed DNA polymerase thermal stability mutant as described above.
[0009] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2 is SEQ ID NO. 1; and the gene encoding the rationally designed DNA polymerase thermal stability mutant is obtained by single point mutation based on the nucleotide sequence shown in SEQ ID NO. 1.
[0010] Preferably, the nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 are SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, respectively.
[0011] The present application provides a recombinant plasmid comprising the gene as described above.
[0012] The present application provides a soluble protein, immobilized enzyme or engineered bacteria comprising the rationally designed DNA polymerase thermal stability mutant or gene as described above.
[0013] The present application provides a construction method of the rationally designed DNA polymerase thermal stability mutant as described above, comprising the following steps: By searching the amino acid sequence shown in SEQ ID NO. 2 in the Pfam database and the NCBI database, removing the same sequence that appears repeatedly, selecting the amino acid sequence with more than 30% identity with the amino acid sequence shown in SEQ ID NO. 2, and then performing multiple sequence alignment by Clustalx v1.83 software, the remaining amino acid sequence is arranged into a fasta. file uploaded to the Consensus Maker v2.0.0 server, and the online software will generate a consensus sequence that can be edited later after modifying the parameters as needed; By AlphaFold online tool for predicting the three-dimensional structure of the protein shown in SEQ ID NO. 2, PyMOL is used to observe the crystal structure of the protein shown in SEQ ID NO. 2, and then RoseTTA Fold3.14 is used to predict and screen out the mutation sites related to thermal stability as follows: H149D, T203D, R306I, N313P, K422E.
[0014] Preferably, the amplification primer sequence of the DNA polymerase heat stability mutant gene encoding the mutation site H149D is SEQ ID NO. 10, SEQ ID NO. 11; The amplification primer sequence of the DNA polymerase heat stability mutant gene encoding the mutation site T203D is SEQ ID NO. 12, SEQ ID NO. 13; The amplification primer sequence of the DNA polymerase heat stability mutant gene encoding the mutation site R306I is SEQ ID NO. 14, SEQ ID NO. 15; The amplification primer sequence of the DNA polymerase heat stability mutant gene encoding the mutation site N313P is SEQ ID NO. 16, SEQ ID NO. 17; The amplification primer sequence of the DNA polymerase heat stability mutant gene encoding the mutation site K422E is SEQ ID NO. 18, SEQ ID NO. 19.
[0015] The application provides the application of the DNA polymerase heat stability mutant based on rational design in DNA amplification.
[0016] The application has the following advantages: 1. The DNA polymerase heat stability mutant based on rational design provided by the application is a single-point mutant, and compared with the wild-type Phi29 DNA polymerase, the single-point mutant has better thermal stability at 40-70 DEG C.
[0017] 2.The application provides a construction method of a DNA polymerase thermal stability mutant based on rational design, which is different from rational design based on the precise structure-function relationship of a protein, and is guided by the Consensus Concept theory, analyzes information capable of improving enzyme thermal stability from an evolutionary perspective, performs integrated analysis on a Phi29 DNA polymerase sequence by RoseTTA Fold 3.14, and obtains a new Phi29 DNA polymerase mutant with high stability by combining bioinformatics and crystallography methods.
[0018] 3.The DNA polymerase thermal stability mutant based on rational design provided by the application has excellent catalytic activity and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 The wild-type Phi29 DNA polymerase protein simulation crystal structure schematic diagram and the distribution of the mutation site on the crystal structure schematic diagram are provided for the embodiment 2 of the application. DETAILED DESCRIPTION
[0020] The application will be further described in detail below with reference to the embodiments, so that those skilled in the art can implement the application according to the description.
[0021] It should be understood that the terms such as “have”, “contain” and “include” used herein do not exclude the presence or addition of one or more other elements or combinations thereof.
[0022] Unless otherwise specified, the test methods used in the following examples are all conventional methods. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial channels. Unless otherwise specified, the specific conditions in the following examples are carried out according to conventional conditions or manufacturer's recommended conditions. Unless otherwise specified, the reagents or instruments used are conventional products that can be obtained by commercial purchase.
[0023] Example 1 The embodiment provides a DNA polymerase thermal stability mutant based on rational design (which can also be referred to as a Phi29 DNA polymerase mutant), wherein the Phi29 DNA polymerase is derived from a Bacillus subtilis bacteriophage, the wild-type Phi29 DNA polymerase is named Phi29 Pol, the nucleic acid sequence encoding the Phi29 DNA polymerase protein is SEQ ID NO. 1, and the amino acid sequence is SEQ ID NO. 2.
[0024] The DNA polymerase thermostability mutants based on rational design provided in this embodiment include: forming a derivative protein with the same function as the amino acid sequence shown in SEQ ID NO.2 (i.e., Phi29 DNA polymerase protein) by substituting, deleting, or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.2; or forming a derivative protein with at least 90% homology to the amino acid sequence shown in SEQ ID NO.2 (i.e., Phi29 DNA polymerase protein) by substituting, deleting, or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.2.
[0025] Specifically, single-point mutations were performed at specific sites on the amino acid sequence shown in SEQ ID NO.2 to obtain five single-point mutants of Phi29 DNA polymerase. The mutation sites are: H149D, T203D, R306I, N313P, and K422E. After activity determination, their amino acid sequences correspond to SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. The nucleotide sequences encoding these amino acid sequences are SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively.
[0026] Example 2 This embodiment provides a method for constructing a DNA polymerase thermostability mutant based on rational design, including the following steps: 1. Cloning of the wild-type Phi29 DNA polymerase gene The wild-type Phi29 DNA polymerase gene was codon optimized using Escherichia coli as the host cell to obtain the optimized Phi29 DNA polymerase gene, whose nucleic acid sequence is SEQ ID NO.1 and the expressed amino acid sequence is SEQ ID NO.2. Using SEQ ID NO.1 as the target gene, the target gene was amplified using upstream amplification primer SEQ ID NO.8 and downstream amplification primer SEQ ID NO.9. The nucleic acid sequence of SEQ ID NO.8 is: 5'-TGGTGATGATGGTGTCCCTTGATGGTGA-3'; The nucleic acid sequence of SEQ ID NO.9 is: 5'-GAAGGAGATATACATATGAAGCACATG-3'.
[0027] The amplification conditions are: pre-denaturation at 98°C for 30 s, amplification at 98°C for 10 s, then amplification at 60°C for 10 s, amplification at 72°C for 40 s, a total of 35 cycles, and finally amplification at 72°C for 5 min.
[0028] After the reaction was completed, 1% agarose gel electrophoresis was used to detect the PCR amplification product, and a 7.0 kb band was obtained, which was consistent with the expected result. According to the standard operation of the kit, the target fragment was recovered and purified, the target fragment and the pET-21a(+) plasmid were double-digested using restriction endonucleases Xhol and Ndel, and then T4 DNA ligase was used for ligation. The obtained ligation product was transformed into E. coli BL21(DE3) competent cells, the transformed cells were plated on LB plates containing 50 μg / mL ampicillin, the positive clone plasmid was extracted, and sequencing was performed. The results showed that the cloned Phi29 DNA polymerase gene sequence was correct, and it had been correctly inserted into the pET-21a(+) plasmid, obtaining the recombinant plasmid pET21a-Phi29; The wild-type Phi29 DNA polymerase is derived from Bacillus subtilis phage Phi29. Bacillus subtilis The Phi29 DNA polymerase gene is provided by Suzhou Jinyuzhi Biotechnology Co., Ltd. The PCR amplification enzyme is PrimeSTAR Max DNA Polymerase provided by Takara.
[0029] 2. Expression and purification of Phi29 DNA polymerase protein The engineering bacteria in the glycerol tube were inoculated into 4 mL LB medium test tubes containing 100 μg / mL Amp + at a volume ratio of 1%, and cultured at 37°C, 220 rpm for 12 h. 4 mL of bacterial solution was transferred to a 1 L LB medium flask containing 50 μg / mL Amp + , and cultured at 37°C, 220 rpm for 2.5 h to make OD600 reach about 0.9. The final concentration of IPTG inducer was 0.1 mM, and the culture was induced at 25°C, 200 rpm for 14 h. The harvested E. coli cell suspension after fermentation was ultrasonically broken, and then subjected to one-step Ni-NTA affinity chromatography to obtain Phi29 DNA polymerase protein with a purity of >95%, and the amino acid sequence was SEQ ID NO. 2.
[0030] 3. Multiple sequence alignment and consensus analysis of Phi29 DNA polymerase homologous proteins 3.1. Enter the homepage of Pfam database (http: / / pfam.xfam.org / ), input the amino acid sequence of Phi29 DNA polymerase in the SEQUENCE SEARCH tool to search, the server will directly feedback the alignment results of the amino acid sequences of the entire family of the protein, and the abundance of various amino acids at each mutation site will be displayed in the form of a column chart. This website can also automatically generate the consensus sequence of the protein family; 3.2. Input the amino acid sequence shown in SEQ ID NO. 2 into the NCBI protein database and the Pfam database, and use the Blast tool to find all protein sequences with more than 30% identity to the amino acid sequence of Phi29 DNA polymerase protein (SEQ ID NO. 2). Delete the same sequences that appear repeatedly, and arrange the remaining amino acid sequences into fasta. format, and input them into Clustalx v1.83 software for multiple sequence alignment. The alignment results are output in aln., dnd. and fasta. formats. The dnd. file is an evolutionary tree file, and the aln. and fasta. files are sequence files in different formats. Upload the above fasta. file to the Consensus Maker v2.0.0 (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html) server, and modify the parameters as needed. The online software will generate a consensus sequence that can be edited later.
[0031] 3.3. Compare the amino acid sequence of Phi29 DNA polymerase protein (SEQ ID NO. 2) with the consensus sequence of the family and the amino acid abundance chart at each site.
[0032] 4. Simulation of the three-dimensional structure of Phi29 DNA polymerase protein and selection of mutation hotspots 4.1. Obtain the three-dimensional structure prediction of Phi29 DNA polymerase protein (amino acid sequence SEQ ID NO. 2) through AlphaFold online tool; 4.2. Observe the crystal structure of Phi29 DNA polymerase protein (amino acid sequence SEQ ID NO. 2) with PyMOL, and then predict and screen mutation sites related to thermal stability through RoseTTA Fold 3.14. According to the structure information, review the above-mentioned mutation sites and mutation forms, and screen the mutation site that is most likely to improve the thermal stability of Phi29 DNA polymerase protein. The screening conditions are as follows: (1) The criteria for determining a site as a candidate site are: ① The overall amino acid abundance of most proteins in the family at this site is high; ② The amino acid at this site is conserved; ③ The amino acid with a high frequency at this site has a large physicochemical property difference with the amino acid at this site in the Phi29 DNA polymerase protein, such as charge difference, polarity strength, steric hindrance size, etc.
[0033] (2) Remove the amino acid residues near the active center, i.e. within 10 Å of the catalytic residues, except for the amino acid residues in the embedded or semi-embedded state.
[0034] After the above two steps of screening, a total of 9 difference sites remain, most of which are located on the surface of the Phi29 DNA polymerase protein molecule.
[0035] (3) According to the crystal structure of Phi29 DNA polymerase, the above 9 mutant forms are analyzed in detail one by one to screen out mutants that can improve the thermal stability of Phi29 DNA polymerase protein.
[0036] The main criteria for judgment are: ① The mutation should eliminate the original form of force that is not conducive to thermal stability, such as electrostatic repulsion, charge aggregation, etc.; ② The mutation should not destroy the existing form of force that is conducive to thermal stability and stable protein structure; ③ The mutation should introduce new forms of force that are conducive to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interactions, etc.
[0037] A total of 5 single-point mutants were designed, with mutation sites at H149D, T203D, R306I, N313P, and K422E. The 5 Phi29 DNA polymerase single-point mutants were subjected to activity determination, and 5 Phi29 DNA polymerase mutants with improved thermal stability were screened, with mutation sites at H149D, T203D, R306I, N313P, and K422E, and the corresponding single-point mutant amino acid sequences were SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7.
[0038] 5. Construction, expression, and purification of mutants 5.1. Construction of DNA polymerase thermal stability mutants based on rational design The recombinant plasmid pET21a-Phi29 in step 1 is used as a template, a pair of complementary oligonucleotides with mutation sites are used as amplification primers, and PrimeSTAR Max DNA Polymerase high-fidelity enzyme is used for whole plasmid PCR amplification to obtain a recombinant plasmid with specific mutation sites; The amplification primer pair used is: (1) The nucleic acid sequences of the upstream amplification primer SEQ ID NO. 10 and the downstream amplification primer SEQ ID NO. 11 of the mutation site H149D are as follows: SEQ ID NO. 10: 5'-CGACTACgacAAAGAACGCCCGGTTGGTTATAAGAT-3'; SEQ ID NO. 11: 5'-CGTTCTTTgtcGTAGTCGATGTCGCCCTTCAGAACC-3'; (2) The nucleic acid sequences of the upstream amplification primer SEQ ID NO. 12 and the downstream amplification primer SEQ ID NO. 13 of the mutation site T203D are as follows: SEQ ID NO. 12: 5'-CATCATCgacACCAAGAAGTTCAAGAAGGTGTTCCCGAC-3'; SEQ ID NO. 13: 5'-TCTTGGTgtcGATGATGTCCTTGAAACCTTTCAGGCTGTC-3'; (3) The nucleic acid sequences of the upstream amplification primer SEQ ID NO. 14 and the downstream amplification primer SEQ ID NO. 15 of the mutation site R306I are as follows: SEQ ID NO. 14: 5'-CAAATCAAGatcAGCCGTTTCTATAAAGGCAATGAGTATCTG-3' SEQ ID NO. 15 5'-GAAACGGCTgatCTTGATTTGAATCGTCGGAATGTAGCC-3' (4) The nucleic acid sequences of the upstream amplification primer SEQ ID NO. 16 and the downstream amplification primer SEQ ID NO. 17 of the mutation site N313P are as follows: SEQ ID NO. 16: 5'-CTATAAAGGCccaGAGTATCTGAAGAGCAGCGGTGG-3' SEQ ID NO.17 5'-CAGATACTCtggGCCTTTATAGAAACGGCTGCGC-3' (5) The nucleic acid sequences of the upstream amplification primer SEQ ID NO. 19 and the downstream amplification primer SEQ ID NO. 20 of the mutation site K422E are as follows, respectively: SEQ ID NO.18 5'-GAAGAAACCgaaGATCCGGTGTATACCCCAATGGG-3' SEQ ID NO.19 5'-CACCGGATCttcGGTTTCTTCCTCGCCTAAACGG-3' The amplification conditions are: pre-denaturation at 98℃ for 30 s, amplification at 98℃ for 10 s, then amplification at 60℃ for 10 s, amplification at 72℃ for 40 s, a total of 35 cycles, and finally amplification at 72℃ for 5 min.
[0039] The PCR amplification product is recovered by gel, and the recovered product is digested with DpnI enzyme at 37℃ for 1 h to degrade the original template; the digestion product is transformed into E. coli BL21(DE3) competent cells, spread on LB agar plates containing 50 μg / mL Amp resistance, incubated at 37℃ overnight, positive clones are selected, and sequencing verification is performed to obtain recombinant bacteria containing Phi29 DNA polymerase single-point mutant; The PCR amplification enzyme is PrimeSTAR Max DNA Polymerase provided by Takara.
[0040] The above DpnI enzyme is provided by TakaRa company.
[0041] Test example DNA polymerase thermostability mutant The wild-type Phi29 DNA polymerase and the various NA polymerase thermostability mutants provided in Example 2 are subjected to thermostability test, according to the conventional Phi29 DNA polymerase activity determination method, specifically: Incubate the enzyme solution at different temperatures for 30 min, sample at the same treatment time, and determine the residual activity percentage of Phi29 DNA polymerase or Phi29 DNA polymerase mutant; The test results are shown in Table 1: Table 1 Enzymatic property characterization results of wild-type Phi29 DNA polymerase and single-point mutant As shown in Table 1, compared with wild-type Phi29 DNA polymerase, the five DNA polymerase thermal stability mutants provided by the present application have better thermal stability at 40-70°C.
[0042] Although the embodiments of the present application have been disclosed as above, they are not limited only to the application listed in the specification and the embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and thus the present application is not limited to specific details, without departing from the general concept defined by the claims and the equivalent scope.
Claims
1. A DNA polymerase thermostability mutant based on rational design, characterized in that, DNA polymerase thermostability mutants include single-point mutants with any one of the single-point mutation sites H149D, T203D, R306I, N313P, or K422E in the amino acid sequence shown in SEQ ID NO.
2.
2. The DNA polymerase thermostability mutant based on rational design according to claim 1, characterized in that, The amino acid sequences of the DNA polymerase thermostability mutants obtained by mutation sites H149D, T203D, R306I, N313P, and K422E are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively.
3. A gene encoding a rationally designed DNA polymerase thermostability mutant as described in any one of claims 1-2.
4. The gene according to claim 3, characterized in that, The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 is SEQ ID NO.1; the genes encoding the rationally designed DNA polymerase thermostability mutants are all obtained by single-point mutation based on the nucleotide sequence shown in SEQ ID NO.
1.
5. The gene according to claim 3, characterized in that, The nucleotide sequences of the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 are SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, respectively.
6. A recombinant plasmid comprising the gene as described in any one of claims 3-5.
7. A soluble protein, immobilized enzyme, or engineered bacterium comprising a rationally designed thermostable mutant of DNA polymerase as described in any one of claims 1-2 or a gene as described in any one of claims 3-5.
8. A method for constructing a DNA polymerase thermostable mutant based on rational design as described in any one of claims 1-2, characterized in that, Includes the following steps: By searching the Pfam and NCBI databases for the amino acid sequence shown in SEQ ID NO.2, redundant amino acid sequences were removed, and amino acid sequences with greater than 50% identity with the amino acid sequence shown in SEQ ID NO.2 were selected. Then, multiple sequence alignment was performed using Clustalx v1.83 software, and the remaining amino acid sequences were compiled into a FASTA file and uploaded to the Consensus Maker v2.0.0 server. After modifying the settings as needed, the online software will generate a consensus sequence that can be edited later. The three-dimensional structure of the protein in SEQ ID NO.2 was predicted using the AlphaFold online tool, and then stability-related mutation sites were screened out using RoseTTAFold 3.14: H149D, T203D, R306I, N313P, and K422E.
9. The construction method according to claim 8, characterized in that, The amplification primer sequences for the gene encoding a thermostable mutant of DNA polymerase with mutation site H149D are SEQ ID NO.10 and SEQ ID NO.11; The primer sequences for amplifying the gene encoding a DNA polymerase thermostable mutant with the mutation site T203D are SEQ ID NO.12 and SEQ ID NO.13; The primer sequences for amplifying the gene encoding a thermostable mutant of DNA polymerase with mutation site R306I are SEQ ID NO.14 and SEQ ID NO.15; The primer sequences for amplifying the gene encoding a thermostable mutant of DNA polymerase with the mutation site N313P are SEQ ID NO.16 and SEQ ID NO.
17. The amplification primer sequences for the gene encoding the thermostable mutant of DNA polymerase with mutation site K422E are SEQ ID NO.18 and SEQ ID NO.
19.
10. The application of the rationally designed thermostable mutant of DNA polymerase as described in any one of claims 1-2 in DNA amplification.