CN-122012448-A - Stable high-efficiency high-fidelity DNA polymerase mutant and preparation method thereof

Abstract

The invention discloses a stable high-efficiency high-fidelity DNA polymerase mutant and a preparation method thereof, which relate to the fields of genetic engineering and minute biology, wherein the high-fidelity DNA polymerase is modified by rationally designing mutation sites and optimizing additives, so that the thermal stability, the extension efficiency and the continuous synthesis capability of the high-fidelity DNA polymerase are enhanced, and meanwhile, the high-fidelity characteristics of the high-fidelity DNA polymerase are maintained. The optimized DNA polymerase has obviously improved amplification speed in the PCR amplification process, can efficiently synthesize longer DNA fragments, and is suitable for the amplification of complex templates (such as sequences with high GC content or abundant secondary structures). The method remarkably improves the efficiency and accuracy of long fragment DNA synthesis, and provides a more efficient tool for genome sequencing, gene cloning, in-vitro diagnosis and the like.

Inventors

• LIU XIAOMING
• LIU JIALU
• LV RONGWEI
• ZHANG RUIJUAN
• CHEN YIYING
• ZHANG LIN

Assignees

• 优基生物科技(杭州)有限公司

Dates

Publication Date

20260512

Application Date

20260128

Claims (10)

1. 1. A stable high-efficiency high-fidelity DNA polymerase mutant, which is characterized in that the stable high-efficiency high-fidelity DNA polymerase mutant is subjected to one or more mutations in the following sites on the basis of a wild Pfu DNA polymerase, wherein the mutation point is V93M, V93K, V5393L, R97E, R379H, I522H.
2. 2. The stable high-efficiency high-fidelity DNA polymerase mutant of claim 1, wherein the amino acid sequence of the stable high-efficiency high-fidelity DNA polymerase mutant is shown in SEQ ID No. 1-12.
3. 3. The stable high-efficiency high-fidelity DNA polymerase mutant according to claim 1 or2, wherein the nucleotide sequence of the stable high-efficiency high-fidelity DNA polymerase mutant is shown in SEQ ID No. 13-24.
4. 4. The stable high-efficiency high-fidelity DNA polymerase mutant according to claim 1, wherein the amino acid sequence of the wild-type Pfu DNA polymerase is shown in SEQ ID No. 25.
5. 5. The stable high-efficiency high-fidelity DNA polymerase mutant according to claim 1, wherein the stable high-efficiency high-fidelity DNA polymerase mutant comprises three site mutations V93M, R379H, I H, the amino acid sequence of which is shown in SEQ ID No. 8.
6. 6. A recombinant plasmid comprising a vector and the nucleotide sequence of the stable, high-efficiency, high-fidelity DNA polymerase mutant of claim 3.
7. 7. The recombinant plasmid of claim 6, wherein said vector is a pET-24a (+) plasmid.
8. 8. A method for preparing a stable high-efficiency high-fidelity DNA polymerase mutant, which is characterized in that the stable high-efficiency high-fidelity DNA polymerase mutant according to claims 1-5 is prepared, comprising the following steps: S1, introducing mutation by taking a wild pfu gene as a template; S2, cloning the mutant gene into a pET-28a vector to transform E.coli BL21 (DE 3); s3, after IPTG induction expression, obtaining mutant enzyme through purification.
9. 9. The preparation method of the stable and high-efficiency high-fidelity DNA polymerase mutant is characterized in that mutation points in S1 are arranged in the middle of a primer, two PCR amplifications are carried out after mutation is introduced in S1, S2 comprises double enzyme digestion, connection, transformation and screening, the induction temperature of S3 is 16-25 ℃, the induction time is 10-20h, the induction reagent is IPTG reagent, and the purification in S3 is nickel column purification.
10. 10. The method for preparing a stable and efficient high-fidelity DNA polymerase mutant according to claim 9, wherein the conditions of the double-cutting enzyme in the step S2 are 37 ℃ for 1-3h, and the conditions of the connection are 16 ℃ for 12-16h.

Description

Stable high-efficiency high-fidelity DNA polymerase mutant and preparation method thereof Technical Field The invention relates to the fields of genetic engineering and molecular biology, in particular to a stable high-efficiency high-fidelity DNA polymerase mutant and a preparation method thereof. Background Polymerase Chain Reaction (PCR) technology plays a vital role in the fields of molecular biology, medical diagnosis, gene synthesis, synthetic biology, and the like. Among them, high-fidelity DNA polymerase is the first choice tool for high-precision gene amplification, mutation detection and long fragment cloning due to its extremely low mismatch rate in the PCR process. Currently, commonly used high-fidelity polymerases Pfu enzyme, KOD enzyme, vent enzyme, phusion enzyme, etc. generally have 3'→5' exonuclease activity, and can correct mismatched bases, thereby significantly reducing mutation rate in PCR process. However, these high fidelity enzymes still have some problems in practical applications, mainly including limited amplification of long fragments, low amplification efficiency of high GC or complex templates, low amplification speed, etc. Therefore, a novel DNA polymerase having high fidelity, rapid amplification capability and long fragment amplification efficiency needs to be developed to meet the application requirements in the fields of high throughput sequencing, large fragment gene assembly, precise medicine and the like. Patent document CN114574464B discloses a high-fidelity DNA polymerase mutant and application thereof, wherein the DNA polymerase mutant has a defect of insufficient anti-inhibition capability and poor thermal stability. Patent document CN120624397a discloses a high-fidelity Pfu DNA polymerase mutant, a preparation method and application thereof, wherein the DNA polymerase mutant has slower amplification efficiency and insufficient anti-inhibition capability. In summary, there is a need for an improved anti-repression capability, improved thermal stability, and at the same time high fidelity, rapid amplification capability, and long fragment amplification capability. Disclosure of Invention The invention aims to provide a stable and high-efficiency high-fidelity DNA polymerase mutant, which is characterized in that one or more mutations of V93M, V93K, V93L, R97E, R379H, I522H are carried out on the basis of wild Pfu DNA polymerase, so that the inhibition resistance and the thermal stability of the DNA polymerase mutant are improved, and the amplification speed and the long fragment amplification performance are further improved. It is another object of the present invention to provide a high-fidelity DNA polymerase with mutations at three sites, the mutation sites being V93M, A379H, I522H. It is yet another object of the present invention to provide a PCR reaction buffer that is effective to promote the induction of mutations to form DNA polymerase mutants. The invention also aims to provide a preparation method of the high-fidelity DNA polymerase mutant, which can simply and accurately prepare the DNA polymerase mutant. In order to achieve the above purpose, the invention discloses a high-fidelity DNA polymerase mutant, which is characterized in that one or more mutations in the following points are carried out on the basis of wild Pfu DNA polymerase, wherein the mutation points are V93M, V93K, V L, R97E, A379H, I522H; the V93M mutation means that valine (Val) at the 93 th site is mutated into methionine (Met), substitution between hydrophobic amino acids, the side chain of methionine is longer, more flexible and contains sulfur atoms, local flexibility is increased, stability at high temperature is improved, influence on enzyme activity is small, valine (Val) at the 93 th site is mutated into lysine (Lys), hydrophobic amino acids are changed into positively charged hydrophilic amino acids, electrostatic interaction with a DNA skeleton is enhanced, binding affinity of DNA is improved, structure dynamic property is increased, valine (Val) at the 93 th site is mutated into leucine (Leu), both are hydrophobic amino acids, side chains of leucine are slightly larger, local hydrophobic interaction is increased, stability of structure is improved, arginine (Arg) at the 97 th site is mutated into glutamic acid (Glu), positive charge is changed from positive charge, nonspecific binding is reduced after mutation, specificity is improved, local charge environment is changed, conformation is influenced, an effect of 'electrostatic capture' is reduced for polymerase, fidelity is improved, arginine (Arg) at the 93 th site is mutated into histidine (His), protonic arginine (His) can be changed from positive charge to histidine, physiological efficiency is improved or physiological efficiency is improved, the isoleucine (Ile) on the 522 # point is mutated into histidine (His), which is changed from water delivery into polar and protonatable histidine, and new hydrogen bond is introduc