Stable, high-efficiency and high-fidelity DNA polymerase mutant and preparation method thereof

Abstract

The invention discloses a stable and high-efficiency high-fidelity DNA polymerase mutant and a preparation method thereof, relates to the fields of genetic engineering and minute biology, and aims to modify a high-fidelity DNA polymerase by rationally designing mutation sites and optimizing additives, enhance the thermal stability, extension efficiency and continuous synthesis capability of the high-fidelity DNA polymerase and maintain the high-fidelity characteristic of the high-fidelity DNA polymerase. The optimized DNA polymerase shows significantly improved amplification speed in the PCR amplification process, can efficiently synthesize longer DNA fragments, and is suitable for amplification of complex templates (such as sequences with high GC content or rich secondary structures). According to the method, the synthesis efficiency and accuracy of the long-fragment DNA are remarkably improved, and a more efficient tool is provided for genome sequencing, gene cloning, in-vitro diagnosis and the like.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular biology, specifically to a stable and efficient high-fidelity DNA polymerase mutant and its preparation method. Background Technology

[0002] Polymerase chain reaction (PCR) technology plays a crucial role in molecular biology, medical diagnostics, gene synthesis, and synthetic biology. Among them, high-fidelity DNA polymerases, due to their extremely low mismatch rate during PCR, have become the preferred tool for high-precision gene amplification, mutation detection, and long-fragment cloning.

[0003] Currently, commonly used high-fidelity polymerases such as Pfu, KOD, Vent, and Phusion typically possess 3'→5' exonuclease activity, enabling them to correct mismatched bases and significantly reduce the mutation rate during PCR. However, these high-fidelity enzymes still face some challenges in practical applications, primarily including limitations in amplifying long fragments, low amplification efficiency with high GC or complex templates, and slow amplification speed. Therefore, there is a need to develop a novel DNA polymerase that combines high fidelity, rapid amplification capability, and long fragment amplification efficiency to meet the application requirements of high-throughput sequencing, large-fragment genome assembly, and precision medicine.

[0004] Patent document CN114574464B discloses a high-fidelity DNA polymerase mutant and its application. However, the DNA polymerase mutant has insufficient resistance to inhibition and poor thermal stability.

[0005] Patent document CN120624397A discloses a high-fidelity Pfu DNA polymerase mutant, its preparation method, and its applications. However, the DNA polymerase mutant exhibits slow amplification efficiency and insufficient resistance to inhibition. Therefore, there is a need for a method that improves resistance to inhibition, enhances thermal stability, and simultaneously possesses high fidelity, rapid amplification capability, and long-fragment amplification performance. Summary of the Invention

[0006] One objective of this invention is to provide a stable and highly efficient high-fidelity DNA polymerase mutant, which is based on wild-type Pfu DNA polymerase and undergoes one or more mutations in V93M, V93K, V93L, R97E, R379H, and I522H, thereby improving the DNA polymerase mutant's resistance to inhibition and thermal stability; and further improving amplification speed and long fragment amplification performance.

[0007] Another objective of this invention is to provide a high-fidelity DNA polymerase with three mutated sites: V93M, A379H, and I522H.

[0008] Another objective of this invention is to provide a PCR reaction buffer that effectively promotes the formation of DNA polymerase mutants through induced mutations.

[0009] Another objective of this invention is to provide a method for preparing a high-fidelity DNA polymerase mutant, which can simply and accurately prepare the DNA polymerase mutant.

[0010] To achieve the above objectives, this invention discloses a high-fidelity DNA polymerase mutant. Based on wild-type Pfu DNA polymerase, one or more mutations are performed at the following sites: V93M, V93K, V93L, R97E, A379H, and I522H. The V93M mutation represents a change from valine (Val) at site 93 to methionine (Met). This substitution between hydrophobic amino acids results in a longer, more flexible side chain containing sulfur atoms, increasing local flexibility and improving stability at high temperatures with minimal impact on enzyme activity. The V93 mutation also represents a change from valine (Val) to lysine (Lys), transforming a hydrophobic amino acid into a positively charged hydrophilic amino acid, enhancing electrostatic interactions with the DNA backbone, increasing DNA binding affinity, and improving structural dynamics. Finally, the V93 mutation also represents a change from valine (Val) to leucine (Lys). Both leucine and glutamic acid are hydrophobic amino acids. Leucine has a slightly larger side chain, which increases local hydrophobic interactions and improves structural stability. Arginine (Arg) at position 97 is mutated to glutamic acid (Glu), changing from positively charged to negatively charged. This mutation reduces non-specific binding, increases specificity, changes the local charge environment, and affects conformation, thus reducing the "electrostatic trapping" effect of polymerase and improving fidelity. Arginine (Arg) at position 379 is mutated to histidine (His), changing from positively charged arginine to protonable histidine. Histidine may be positively or negatively charged at physiological pH, regulating catalytic efficiency, improving fidelity, and making enzyme activity more optimal at specific pH. Isoleucine (Ile) at position 522 is mutated to histidine (His), changing from water-transporting to polar, protonable histidine, introducing new hydrogen bonds, changing substrate specificity or catalytic mechanism, and improving stability.

[0011] As a preferred embodiment, the amino acid sequence of the high-fidelity DNA polymerase mutant is shown in SEQ ID NO.1-12. The simultaneous occurrence of the three mutations V93M, R97E, and R379H yields a polymerase with high thermostability and high fidelity. V93M affects thermostability, R97E improves specificity, and R379H regulates catalytic efficiency. V93K, I522H, and R97E primarily affect the affinity between the polymerase and the template, increasing the extension length and speed, making it suitable for long fragment amplification. R379H and I522H yield a polymerase with high fidelity, high enzyme catalytic activity, and high stability. V93L, R97E, and I522H yield a polymerase that improves amplification speed, product length, and catalytic properties.

[0012] As a preferred embodiment, the nucleotide sequence of the high-fidelity DNA polymerase mutant is shown in SEQ ID NO.13-24.

[0013] As a preferred option, the amino acid sequence of wild-type Pfu DNA polymerase is shown in SEQ ID NO: 25.

[0014] Preferably, the high-fidelity DNA polymerase mutant contains mutations at three sites: V93M, R379H, and I522H, with the amino acid sequence shown in SEQ ID NO: 9. V93M slightly enhances local hydrophobic stacking, improving thermal stability and structural rigidity.

[0015] A recombinant plasmid, characterized in that the recombinant plasmid comprises a vector and the nucleotide sequence of the high-fidelity DNA polymerase mutant.

[0016] Preferably, the vector is the pET-24a(+) plasmid.

[0017] A method for preparing a high-fidelity DNA polymerase mutant includes the following steps: S1. Introduce mutations using the wild-type pfu gene as a template; S2. The mutant gene was cloned into the pET-28a vector and transformed into E.coli BL21 (DE3). After S3 and IPTG induction, mutant enzymes were obtained through purification.

[0018] Preferably, the mutation point in S1 is located in the middle of the primer; after the mutation is introduced in S1, two PCR amplifications are performed; S2 includes double enzyme digestion, ligation, transformation and screening; the induction temperature of S3 is 16-25℃, the induction time is 10-20h, and the induction reagent is IPTG reagent; the purification in S3 is nickel column purification.

[0019] Preferably, the double digestion enzyme in S2 is cultured at 37°C for 1-3 hours; the ligation is performed at 16°C for 12-16 hours.

[0020] The beneficial effects of this invention are: The present invention provides a high-fidelity DNA polymerase mutant, which is obtained by mutating multiple amino acids (V93M, V93K, V93L, R97E, R379H, I522H) of wild-type Pfu DNA, resulting in a DNA polymerase mutant with rapid amplification, high fidelity, strong resistance to inhibition, good thermal stability, and increased product length. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is an agarose gel electrophoresis image showing the amplification test results of DNA polymerase mutants with mutations at three sites (Val93Met, Arg379His, and Ile522His) for different lengths of nucleoside fragments.

[0023] Figure 2 This is an agarose gel electrophoresis image showing the amplification results of the DNA polymerase mutants with mutations at three sites (Val93Met, Arg379His, and Ile522His) in this invention, based on the elongation rate test.

[0024] Figure 3 This is an agarose gel electrophoresis image of the sensitivity amplification test results of the DNA polymerase mutants with mutations at the three sites Val93Met, Arg379His, and Ile522His in this invention.

[0025] Figure 4 This is an agarose gel electrophoresis image of the DNA polymerase mutants with mutations at three sites (Val93Met, Arg379His, and Ile522His) in this invention, showing the results of long fragment amplification tests. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments.

[0027] General Implementation Examples: A wild-type DNA polymerase, with the following amino acid sequence: 1 MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPPYIYA LLRDDSKIEE VKKITGERHG61 KIVRIVDVEK VEKKFLGKPI TVWKLYLEHP QD V PTI R EKV REHPAVVDIF EYDIPFAKRY 121LIDKGLIPME GEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY 181VEVVSSEREM IKRFLRIIRE KDPDIIVTYN GDSDFDFPYLA KRAEKLGIKL TIGRDGSEPK 241MQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAVE 301SGENLERVAK YSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK 361AYERNEVAPN KPSEEEYQ R R LRESYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVS 421PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL 481DYRQKAILKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRK Y I ELVWKELE EKVGFKVLYI 541DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE 601EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK 661LAIYEQITRP IHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE 721YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGLTSWL NIKKS.

[0028] Table 1 shows the mutation sites selected in the early stages for the original amino acid sequence of Pfu DNA polymerase.

[0029] The properties of the mutant Pfu enzyme were verified through the following examples: This example describes the preparation of mutant Pfu type 8; S1. Using the wild-type Pfu gene as a template, a mutation is introduced: S2. The mutant gene was cloned into the pET-28a vector and transformed into E.coli BL21 (DE3). After S3 and IPTG induction, mutant enzymes were obtained through purification.

[0030] S1. Using overlap extension PCR technology, specific primers including the Val93Met mutation site were designed, and the mutation was gradually introduced into the target gene through two rounds of PCR amplification.

[0031] 1. Primer design: Forward primer F1: Contains the Val93Met mutation site. (2) Reverse primer R1: Contains the Ile522His mutation site. (3) Intermediate mutation primer: Design an intermediate primer pair with the Arg379His mutation.

[0032] 2. First round of PCR amplification: Segmented amplification (2) Use F1 and intermediate mutation primers (2) Use intermediate mutation primers and R1.

[0033] Reaction system (50 μL): 1-10 ng wild-type Pfu gene, 1 μL primers (F1, intermediate mutant primers, R1), 4 μL dNTPs, 0.5-1 U high-fidelity polymerase, 5 μL buffer, and ddH2O to 50 μL.

[0034] Reaction procedure: First, set the reaction temperature to 98℃ for 40 seconds, then change it to 60℃ for 15 seconds, then change it to 72℃, with an extension time of 30 seconds per thousand base pairs, then react at 72℃ for 5 minutes, repeat 30 cycles, and finally cool down to 4℃ for storage.

[0035] 3. Second round of PCR amplification: whole genome splicing amplification The first round of PCR products were mixed and used as templates for the second round. All three primers were used in 3 μL, and the amplification reaction was the same as in the first round of PCR.

[0036] S2. The mutant gene was cloned into the pET-28a vector and transformed into E.coli BL21(DE3).

[0037] 1. Reagents: Vector pET-28a (+), purified mutant Pfu gene fragment obtained from insert S1, restriction endonucleases Ndel and Xhol, ligase T4 DNA Ligase, competent E. coli BL21 (DE3) cells, culture media LB liquid medium and LB solid plates (containing 50 μg / mL kanamycin), other reagents (DNA purification kit, agarose gel extraction kit, SOC resuscitation medium). 2. Double enzyme digestion vector and insert fragment Vector digestion system: 1 μg pET-28a plasmid, 2 μL 10×FastDigest buffer, 1 μL Ndel, 1 μL Xhol, and ddH2O to a final volume of 20 μL.

[0038] Insertion fragment digestion system: 0.5-1 μg of purified PCR product, 2 μL of 10×FastDigest buffer, 1 μL of Ndel, 1 μL of Xhol, and ddH2O to make up to 20 μL.

[0039] The reaction conditions were 37℃ and 1-2 hours.

[0040] 3. Purification of enzyme digestion products The enzyme digestion products were separated by agarose gel electrophoresis, the target band (vector approximately 5.4 kb, insert fragment length of Pfu gene) was excised, the DNA was purified using a gel extraction kit, and the length was determined.

[0041] 4. Connection reaction (1) Ligation system: 50 ng of pET-28a vector after enzyme digestion, 3-5 times the amount of the inserted fragment vector after enzyme digestion, 1 μL of 10×T4Ligase buffer, 0.5 μL of T4 DNA Ligase, and ddH2O to make up to 10 μL.

[0042] (2) The above system is connected at 16℃ for 2-4 hours.

[0043] 5. Transformation E. coli BL21(DE3) competent cells were thawed on ice. 5 μL of ligation product was added to 50 μL of competent cells, and the mixture was gently swished to mix. The cells were then incubated on ice for 30 minutes, followed by heat shock at 42°C for 45 seconds. The cells were then quickly returned to ice for 2 minutes. 450 μL of antibiotic-free LB or SOC medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 45 minutes. 100-200 μL of the recovery solution was spread onto LB plates containing kanamycin (50 μg / mL), and the cells were incubated upside down at 37°C for 12-16 hours.

[0044] 6. Colony screening and validation Select 3-5 single colonies of suitable size and inoculate them into LB liquid medium containing Kanamycin, and incubate overnight at 37°C; extract plasmids using a plasmid extraction kit; digest the plasmids with Ndel and Xhol enzymes, and check by electrophoresis whether the expected size of the insert fragment is released; send the plasmids that have been verified by enzyme digestion to sequence, and use T7 primers to verify whether the mutation site and reading frame are correct.

[0045] S3 and IPTG-induced expression, followed by nickel column purification to obtain the mutant enzyme. 1. Reagents and Instruments: Engineered strain: Verified E. coli BL21(DE3) / pET-28a-Pfu (mutant); Antibiotic: Kanamycin (50 mg / mL stock solution); Inducing agent: IPTG reagent; Culture medium: LB or TB liquid medium; Lysis buffer: 20mM Tris-HCl, pH 8.0; 300mM sodium chloride; 10mM imidazole; 1mM PMSF (protease inhibitor). Elution buffer; Nickel column packing: Ni-NTA Agarose / Sepharose; Instruments: shaker, centrifuge, ultrasonic homogenizer, protein electrophoresis system, chromatography column, ultraviolet spectrophotometer.

[0046] 2. Induced expression of engineered bacteria (1) Activation: Pick a single colony from the screening plate and inoculate it into 3-5 mL of LB medium containing Kanamycin. Incubate overnight at 37°C with shaking at 220 rpm. (2) Expanded culture: The overnight bacteria were transferred to fresh LB medium (containing Kanamycin) at a ratio of 1:100 and cultured at 37°C with vigorous shaking until OD. 600 ≈0.6-0.8 (mid-logarithmic growth phase); (3) Induction: Add IPTG to the culture to a final concentration of 0.1-1 mM; lower the temperature: transfer the culture flask to a shaker at 16-25℃ and induce expression for 16-20 hours by low-speed shaking (180 rpm). Low-temperature slow induction helps reduce inclusion body formation and increase the proportion of soluble protein; (4) Collection of bacteria: Centrifuge at 5000×g for 10 minutes at 4℃ to collect the bacterial cells and discard the supernatant. The bacterial cells can be frozen at -20℃ for later use.

[0047] 3. Cell disruption and preparation of crude extract (1) Resuspending the bacterial cells: Add 5-10 mL of pre-cooled lysis buffer per gram of wet bacteria and vortex; (2) Ultrasonic fragmentation: Under ice bath conditions, the material was broken up using an ultrasonic homogenizer. Parameters: Power 200-300 W, 2-second operation, 4-second interval, total time 5-10 minutes, until the bacterial solution becomes clear and the viscosity decreases. (Maintain low temperature throughout the process to avoid sample overheating and protein degradation); (3) Centrifugation: Centrifuge at 4℃ and 12000 ×g for 30 minutes, and collect the supernatant. The precipitate consists of cell debris and inclusion bodies.

[0048] 4. Purification by nickel column affinity chromatography (1) Column preparation: Take an appropriate amount of Ni-NTA packing material and pack it into the chromatography column. Equilibrate the column with 5-10 column volumes (CV) of binding buffer. (2) Sample loading: The supernatant after centrifugation is slowly loaded onto the equilibrated nickel column, and the flow rate is controlled at 0.5-1 mL / min (gravity column). The flow-through liquid is collected. (3) Washing: Wash the column sequentially with washing buffers containing different concentrations of imidazole to remove non-specifically bound contaminating proteins. First wash: 5-10 CV of washing buffer containing 20 mM imidazole; (4) Elution: Elute stepwise (1 mL / tube) with elution buffer containing 250-500 mM imidazole, and collect the elution peak. Elution buffer formulation: 20 mM Tris-HCl (pH 8.0); 300 mM NaCl; 250-500 mM imidazole; (5) Column regeneration and storage: Thoroughly wash the column with 5-10 CV of buffer containing 500 mM imidazole, and then store it at 4°C with 5-10 CV of 20% ethanol.

[0049] 5. Protein Analysis and Preservation (1) SDS-PAGE analysis: SDS-PAGE electrophoresis was performed on samples before induction, after induction, supernatant, flow-through, washing and elution, and Coomassie brilliant blue staining was performed to confirm the expression and purification of the target protein. (2) Concentration determination: The protein concentration in the eluent was determined using the Bradford or BCA method; (3) Desalting / Buffer Replacement: Replace the protein in the storage buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 50% glycerol) using a desalting column or dialysis. (4) Aliquoting and storage: Aliquot the purified protein, add 50% glycerol and store at -20℃; Finally, purified Pfu enzyme containing three site-directed mutations: Val93Met, Arg379His, and Ile522His was obtained.

[0050] The purified Pfu enzyme containing three site-directed mutations, Val93Met, Arg379His, and Ile522His, was amplified and tested for nucleic acid fragments of different lengths. In a 50 μl amplification system, using 100 ng of Human gDNA as a template, target fragments of lengths of 0.5 kb, 1 kb, 2 kb, 3 kb, 4 kb, and 6 kb were amplified.

[0051] Table 2 Primer pair sequences used for different fragments

[0052] The PCR reaction system for testing the amplification of nucleic acid fragments of different lengths is as follows: Reagents: 2 × Buffer 25 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, template 100 ng, dH2O to bring the total to 50 μL; The PCR program was as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 5 sec, 68℃ extension for 20 sec, for a total of 30 cycles; 68℃ final extension for 1 min.

[0053] PCR buffer solution: Tris-HCl or Tris-OAc buffer system 10mM~250mM; KCl or KOAc 1mM~100mM; MgCl2 or MgOAc 1~5mM; sodium alginate, glucuronic acid, acetylgalactosamine polymer, pentosan 1mM~100mM; dNTP 0.1~1mM; BSA or HSA protein 0.01~0.2%; one or more of the surfactants such as Tween-20, NP-40, Triton X-100, etc. 0.01%~0.2%; naturally screened antigen protein that has a synergistic effect with the mutant 1ng / ul~100ng / ul; enzyme factor 1ng / ul~100ng / ul.

[0054] The PCR amplification products were subjected to agarose gel electrophoresis, such as... Figure 1 As shown, lane M represents a DNA molecular weight marker of 15000, and lanes 1-6 represent target fragments of 0.5kb, 1kb, 2kb, 3kb, 4kb, and 6kb, respectively. The results indicate that the PCR buffer system using the high-fidelity DNA polymerase of this invention can effectively amplify DNA fragments of different lengths with good specificity.

[0055] Extension speed amplification test: Table 3 Primer pair sequences

[0056] The PCR reaction for testing extension rate amplification is as follows: Reagents: 2 × Buffer 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, template 10 ng, dH2O to bring the total to 50 μL; The PCR program was as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 5 sec, 68℃ extension for 1 sec / 2 sec / 5 sec, for a total of 30 cycles; 68℃ final extension for 1 min.

[0057] The PCR amplification products were subjected to agarose gel electrophoresis, such as... Figure 2 As shown, lane M represents a DNA molecular weight marker of 15000, and lanes 1-3 represent the target fragments amplified with extension times of 1 sec, 2 sec, and 5 sec, respectively. The results indicate that the PCR buffer system using the high-fidelity DNA polymerase of this invention can accommodate extension speeds as fast as 1 sec and exhibits good amplification performance.

[0058] Sensitivity Amplification Test In a 50 μl amplification system, 10 pg-1 fg of λ-DNA was used as a template to amplify the target fragment of approximately 2 kb in length.

[0059] Table 4 Primer pair sequences

[0060] The PCR reaction for testing extension rate amplification is as follows: Reagents: 2 × Buffer 25 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, template 10 pg-1 fg, dH2O to bring the total to 50 μL; The PCR program was as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 5 sec, 68℃ extension for 1 sec / 2 sec / 5 sec, for a total of 30 cycles; 68℃ final extension for 1 min.

[0061] The PCR amplification products were subjected to agarose gel electrophoresis, such as... Figure 3 As shown, lane M represents a DNA molecular weight of 15000 Marker, and lanes 1-5 represent electrophoresis results with 10 pg-1 fg template added. The results indicate that the PCR buffer system using the high-fidelity DNA polymerase of this invention can stably amplify template up to 1 fg.

[0062] In a 50 μl amplification system, using 100 ng of human gDNA as a template, target fragments of 10 kb and 15 kb in length were amplified.

[0063] Table 5 Primer pair sequences

[0064] Reagents: 2 × Buffer 25 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, template 100 ng, dH2O to bring the total to 50 μL; The PCR program was as follows: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 sec, 55℃ annealing for 5 sec, 68℃ extension for 10 s / kb, for a total of 30 cycles; 68℃ final extension for 1 min.

[0065] The PCR amplification products were subjected to agarose gel electrophoresis, such as... Figure 4 As shown, lane M represents a DNA molecular weight marker of 15000, lane 1 represents a target fragment of approximately 10 kb, and lane 2 represents different target fragments of approximately 15 kb. The results indicate that the PCR buffer system using the high-fidelity DNA polymerase of this invention can effectively amplify long fragments with high yield.

[0066] Through the above-mentioned amplification tests of different nucleic acid fragments, extension rate layer expansion tests, sensitivity amplification tests, and long fragment amplification tests, it can be seen that the high-fidelity DNA polymerase mutant disclosed in this invention enhances thermal stability, anti-inhibition performance, extension efficiency, and continuous synthesis ability, improves amplification speed and product length, and significantly improves the efficiency and accuracy of long fragment DNA synthesis.

Claims

1. A stable and highly efficient high-fidelity DNA polymerase mutant, characterized in that, The stable and efficient high-fidelity DNA polymerase mutant is based on wild-type Pfu DNA polymerase and undergoes one or more mutations at the following sites: V93M, V93K, V93L, R97E, R379H, and I522H.

2. The stable, high-efficiency, high-fidelity DNA polymerase mutant according to claim 1, characterized in that, The amino acid sequence of the stable and efficient high-fidelity DNA polymerase mutant is shown in SEQ ID NO.1-12.

3. The stable, high-efficiency, high-fidelity DNA polymerase mutant according to claim 1 or 2, characterized in that, The nucleotide sequence of the stable and efficient high-fidelity DNA polymerase mutant is shown in SEQ ID NO.13-24.

4. The stable, high-efficiency, high-fidelity DNA polymerase mutant according to claim 1, characterized in that, The amino acid sequence of wild-type Pfu DNA polymerase is shown in SEQ ID NO:

25.

5. The stable, high-efficiency, high-fidelity DNA polymerase mutant according to claim 1, characterized in that, The stable and efficient high-fidelity DNA polymerase mutant contains mutations at three sites: V93M, R379H, and I522H, and its amino acid sequence is shown in SEQ ID NO:

8.

6. A recombinant plasmid, characterized in that, The recombinant plasmid includes a vector and the nucleotide sequence of the stable, high-efficiency, high-fidelity DNA polymerase mutant as described in claim 3.

7. A recombinant plasmid according to claim 6, characterized in that, The vector is pET-24a(+) plasmid.

8. A method for preparing a stable and efficient high-fidelity DNA polymerase mutant, characterized in that, The preparation of the stable, high-efficiency, high-fidelity DNA polymerase mutant according to claims 1-5 includes the following steps: S1. Introduce mutations using the wild-type pfu gene as a template; S2. The mutant gene was cloned into the pET-28a vector and transformed into E.coli BL21 (DE3). After S3 and IPTG induction, mutant enzymes were obtained through purification.

9. The method for preparing a stable and efficient high-fidelity DNA polymerase mutant according to claim 8, characterized in that, The mutation point in S1 is located in the middle of the primer; after the mutation is introduced in S1, two PCR amplifications are performed; S2 includes double enzyme digestion, ligation, transformation and screening; the induction temperature in S3 is 16-25℃, the induction time is 10-20h, and the induction reagent is IPTG reagent; the purification in S3 is nickel column purification.

10. The method for preparing a stable and efficient high-fidelity DNA polymerase mutant according to claim 9, characterized in that, The conditions for the double digestion enzyme in S2 are: culture at 37°C for 1-3 hours; and ligation conditions are: culture at 16°C for 12-16 hours.

Images