CN117126826A - Mutant of high-fidelity Pfu DNA polymerase, preparation method and application thereof - Google Patents

Abstract

The invention discloses a mutant of high-fidelity Pfu DNA polymerase, a preparation method and application thereof, and belongs to the technical field of genetic engineering. The invention provides a mutant of high-fidelity Pfu DNA polymerase, and a coding gene, a recombinant expression vector and a recombinant host of the mutant, wherein the mutant after separation and purification is used as a broad-spectrum fidelity factor and can be used for improving the fidelity of various DNA polymerases; if Taq and Tth DNA polymerase in the DNA polymerase family A, the fidelity of the mutant can be improved by 4-18 times after Pfu DNA polymerase mutant is added; pfu, vent and Kod DNA polymerase in DNA polymerase family B, the fidelity can be improved by 2-6 times after Pfu DNA polymerase mutant is added.

Description

Mutant of high-fidelity Pfu DNA polymerase, preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a mutant of high-fidelity Pfu DNA polymerase, and a preparation method and application thereof.

Background

DNA polymerase fidelity refers to the ability of a DNA polymerase to avoid or correct erroneously inserted nucleotides when replicating a DNA sequence, thereby ensuring that the PCR amplification product sequence is consistent with the template. Taq DNA polymerase and Pfu DNA polymerase, which are common in PCR amplification reactions, belong to DNA polymerase family A and DNA polymerase family B. Family B DNA polymerases typically have 3'-5' exonuclease activity, also known as proofreading activity, which can cleave off the wrong base and reinsert the correct base when a mismatch occurs, and DNA polymerases with fidelity belong essentially to DNA polymerase family B; the DNA polymerase of family a has no 3'-5' exonuclease activity, and therefore the DNA polymerase has no fidelity. For some experiments requiring high precision and high quality, such as molecular cloning, sequencing, site-directed mutagenesis, gene editing and the like, a fidelity DNA polymerase is indispensable, and if a non-fidelity DNA polymerase is used, sequence errors such as mutation, deletion or insertion in an amplified product may occur, which directly affects the accuracy and reliability of the PCR amplified reaction product, thereby affecting the subsequent experimental results and analysis. Therefore, in performing a high-precision and high-quality PCR amplification reaction, the fidelity of the DNA polymerase is very important for the selection of DNA polymerase, and it should be preferred to use a fidelity DNA polymerase such as Pfu DNA polymerase, vent DNA polymerase, KOD DNA polymerase, etc.

The fidelity DNA polymerase can be divided into five distinct domains: the N-terminal domain (residues 1-130, 327-368), the 3'-5' exonuclease activity domain (131-326), the palm domain (369-450 and 501-588), the finger domain (451-500) and the thumb domain (589-775), and the palm domain, the finger domain and the thumb domain together are referred to as the 5'-3' polymerase activity domain, the fidelity of which derives from their 3'-5' exonuclease activity. However, according to the studies, it has been shown (Cline et al,1996;McInerney et al,2014) that the fidelity of the common DNA polymerases Pfu DNA polymerase, vent DNA polymerase and KOD DNA polymerase are limited by themselves, and generally only 4 to 10 times that of Taq DNA polymerase, for example, pfu DNA polymerase has a base mismatch ratio of about 1.3 to 2.0X10 ^-6 The fidelity is 6-10 times of that of Taq DNA polymerase; vent DNA polymeraseThe base mismatch ratio was about 2.8X10 ^-6 The fidelity was 4 times that of Taq DNA polymerase. The non-authentic Taq DNA polymerase can obtain fidelity after adding a small amount of Pfu DNA polymerase with corrective activity, but the fidelity is only 2-4 times of that of the Taq DNA polymerase.

Disclosure of Invention

The invention aims to provide a mutant of high-fidelity Pfu DNA polymerase, a preparation method and application thereof, wherein the mutant can further improve fidelity and reduce base mismatch rate.

The invention provides a mutant of high-fidelity Pfu DNA polymerase, wherein the amino acid sequence of the mutant comprises a 3'-5' exonuclease activity domain and a 5'-3' polymerase domain of the high-fidelity Pfu DNA polymerase, so that the exonuclease activity in the 3'-5' exonuclease activity domain is improved, and the polymerase activity in the 5'-3' polymerase domain is reduced.

Preferably, the amino acid sequence of the mutant comprises a mutation in the 3'-5' exonuclease active domain of a high-fidelity Pfu DNA polymerase: histidine to lysine, mutation occurs in the 5'-3' polymerase domain: glycine is mutated to proline.

Preferably, when the amino acid sequence of the high-fidelity Pfu DNA polymerase is SEQ ID No.2, the following mutations occur in the amino acid sequence of the mutant: H147K and G387P.

Preferably, the amino acid sequence of the mutant is shown as SEQ ID No. 1.

The invention also provides the coding gene of the mutant.

The invention also provides a recombinant vector containing the coding gene.

The invention also provides a recombinant host for expressing the mutant.

Preferably, the system for expressing the mutant by the recombinant host comprises a prokaryotic expression system.

The invention also provides a method for preparing the mutant by using a prokaryotic expression system, which comprises the following steps: connecting the coding genes into a prokaryotic expression vector to obtain a recombinant vector;

transforming bacteria by using the recombinant vector to obtain recombinant bacteria;

inducing the recombinant bacteria to express, wherein the expression product contains the mutant.

The invention also provides application of the mutant in improving the fidelity of DNA polymerase.

The beneficial effects are that: compared with wild Pfu DNA polymerase, the mutant of the high-fidelity Pfu DNA polymerase provided by the invention has the advantages that the 3'-5' exonuclease activity is improved, and the 5'-3' polymerase activity is weakened, so that the high-fidelity is improved, and the mismatch rate is obviously reduced. The Pfu DNA polymerase mutant provided by the invention can be used as a broad-spectrum fidelity factor for improving the fidelity of various DNA polymerases; if Taq and Tth DNA polymerase in the DNA polymerase family A, the fidelity of the mutant can be improved by 4-18 times after Pfu DNA polymerase mutant is added; pfu, vent and Kod DNA polymerase in DNA polymerase family B, the fidelity can be improved by 2-6 times after Pfu DNA polymerase mutant is added.

Drawings

FIG. 1 is an electrophoretogram of Pfu DNA polymerase mutant recombinant expression vector in example 1; wherein: lane 1 is Pfu DNA polymerase mutant recombinant expression vector DNA, lane 2 is Pfu DNA polymerase mutant recombinant expression vector DNA after double digestion, lane M is 1kb DNA Ladder;

FIG. 2 is a graph showing the results of prokaryotic induction and purification of Pfu DNA polymerase mutant in example 2; wherein: lane 1 shows non-induced expression thalli, lane 2 shows induced expression thalli, lane 3 shows a supernatant after thalli disruption, lane 4 shows a Pfu DNA polymerase mutant purified by nickel ion affinity chromatography, lane 5 shows a Pfu DNA polymerase mutant purified by anion exchange chromatography, and lane M shows a 14.4-116kDa non-pre-dyed protein Marker;

FIG. 3 is a graph showing the results of detection of Pfu DNA polymerase mutant 3'-5' exonuclease activity and 5'-3' polymerase activity in example 3;

FIG. 4 is a graph showing the effect of Pfu DNA polymerase mutant on Taq DNA polymerase mismatch rate and fidelity in example 4;

FIG. 5 is a graph showing the effect of Pfu DNA polymerase mutant on mismatch and fidelity of Tth DNA polymerase in example 4;

FIG. 6 is a graph showing the effect of Pfu DNA polymerase mutant on Pfu DNA polymerase mismatch rate and fidelity in example 5;

FIG. 7 is a graph showing the effect of Pfu DNA polymerase mutant on Vent DNA polymerase mismatch and fidelity in example 5;

FIG. 8 is a graph showing the effect of Pfu DNA polymerase mutant on Kod DNA polymerase mismatch and fidelity in example 5.

Detailed Description

The invention provides a mutant of high-fidelity Pfu DNA polymerase, wherein the amino acid sequence of the mutant comprises a 3'-5' exonuclease activity domain and a 5'-3' polymerase domain of the high-fidelity Pfu DNA polymerase, so that the exonuclease activity in the 3'-5' exonuclease activity domain is improved, and the polymerase activity in the 5'-3' polymerase domain is reduced.

The amino acid sequence of the mutant of the present invention preferably comprises a mutation in the 3'-5' exonuclease active domain of a high-fidelity Pfu DNA polymerase: histidine to lysine, mutation occurs in the 5'-3' polymerase domain: mutation of glycine to proline; more preferably, when the amino acid sequence of the high-fidelity Pfu DNA polymerase is SEQ ID No.2, the following mutation occurs in the amino acid sequence of the mutant: H147K and G387P; most preferably, the amino acid sequence of the mutant is shown as SEQ ID No. 1.

The invention also provides the coding gene of the mutant.

When the coding gene is expressed subsequently, preferably, the codon optimization is carried out according to the preference of a host, and as in the embodiment of the invention, the prokaryotic expression system is utilized to express the mutant, so that the coding gene is subjected to the codon optimization of the escherichia coli expression system, and the nucleotide sequence of the optimized coding gene is shown as SEQ ID No. 3.

The invention also provides a recombinant vector containing the coding gene.

The invention is not particularly limited in the type of the basic vector of the recombinant vector, and the basic vector can be eukaryotic expression vector or prokaryotic expression vector, and in the embodiment, the prokaryotic expression vector is taken as an example for explanation, for example, a preferred pET series vector is selected, and more preferred pET-15b is selected. The coding gene described in SEQ ID No.3 is preferably cloned into a prokaryotic expression vector pET-15b, and the cleavage site is preferably Nde I/XhoI.

The invention also provides a recombinant host for expressing the mutant.

The system for expressing the mutant by the recombinant host according to the invention preferably comprises a prokaryotic expression system, preferably using E.coli cells as a host, more preferably E.coli BL21 (DE 3) cells as a host.

The invention also provides a method for preparing the mutant by using a prokaryotic expression system, which comprises the following steps: connecting the coding genes into a prokaryotic expression vector to obtain a recombinant vector;

transforming bacteria by using the recombinant vector to obtain recombinant bacteria;

inducing the recombinant bacteria to express, wherein the expression product contains the mutant.

In the embodiment of the invention, a pET-15b is taken as a basic vector, a gene shown in SEQ ID No.3 is inserted into Nde I/Xho I sites to obtain a recombinant expression vector, and the recombinant expression vector is transformed into an escherichia coli BL21 (DE 3) expression cell to obtain the recombinant host.

The invention is preferably prepared by recombinant expression cell strain induced expression and purification; the method specifically comprises the following steps:

(1) Activating the recombinant expression cell strain of the Pfu DNA polymerase mutant to obtain a recombinant expression cell strain culture;

(2) Inoculating recombinant expression cell strain culture into LB culture solution containing ampicillin according to the ratio of 1:100, and culturing at 35-38 ℃ and 150-180 rpm until bacterial liquid OD ₆₀₀ Reaching 0.5 to 0.65;

(3) Adding IPTG to the bacterial liquid to a final concentration of 0.5-1 mM, and centrifuging to collect bacterial precipitate after induction for 3 hours at 37 ℃;

(4) Re-suspending the thallus sediment obtained in the step (3) by using pre-cooled lysate;

(5) Ultrasonically crushing thalli in an ice bath;

(6) Incubating the crushed thallus liquid in 80 deg.c water bath for 30min, and centrifuging; filtering the obtained supernatant;

(7) Carrying out nickel ion affinity chromatography on the filtrate, and collecting a sample containing target proteins;

(8) And carrying out anion exchange chromatography on the collected target protein-containing sample to obtain Pfu DNA polymerase mutant.

The invention also provides application of the mutant in improving the fidelity of DNA polymerase.

The invention preferably uses purified Pfu DNA polymerase mutant as a fidelity factor, and adds the purified Pfu DNA polymerase mutant into different DNA polymerases according to a certain proportion, so that the fidelity of the DNA polymerase is improved when the PCR amplification reaction is carried out. The DNA polymerase of the invention preferably comprises DNA polymerase in DNA polymerase family A and DNA polymerase family B, wherein the DNA polymerase family A is preferably Taq and Tth DNA polymerase; the DNA polymerase family B is preferably Pfu, vent and Kod DNA polymerase.

When the mutant is added to DNA polymerase family A, taq and Tth DNA polymerase are preferably added at 2.5U with 20-100 ng Pfu DNA polymerase mutant; when the mutant was added to DNA polymerase family B, pfu, vent and Kod DNA polymerase added 5-30 ng Pfu DNA polymerase mutant in 2.5U.

In the present invention, U is an active unit of polymerase, specifically, an amount of enzyme required for incorporating 10nmol of deoxynucleotide into an acid insoluble substance at 74℃for 30 minutes using activated salmon sperm DNA as a template/primer is defined as 1 active unit (U).

In order to further illustrate the present invention, the following examples are provided to describe in detail the mutant of Pfu DNA polymerase with high fidelity, its preparation method and application, but they should not be construed as limiting the scope of the present invention.

Example 1

Establishment of Pfu DNA polymerase mutant genetic engineering strain

1. Synthesis of Pfu DNA polymerase mutant DNA sequence

According to the amino acid sequence SEQ ID No.1 of the Pfu DNA polymerase mutant, the codon of an escherichia coli expression system is optimized to obtain a DNA sequence SEQ ID No.3 capable of being efficiently expressed in escherichia coli, and the DNA sequence SEQ ID No.3 is delivered to a SmaI site of a pUC57 vector by artificially synthesizing the Pfu DNA polymerase mutant by Shanghai biological engineering company.

2. Construction of Pfu DNA polymerase mutant recombinant expression vector

The whole gene sequence of Pfu DNA polymerase mutant is amplified by PCR with pUC57 vector DNA as a template, and the amplification primer sequence is as follows:

PfuF(SEQ ID No.4)：5'-GGAATTCCATATGATTCTGGACGTCGATTATATCAC-3'、

PfuR(SEQ ID No.5)：5'-CCGCTCGAGTTATGATTTCTTGATATTCAGCCACGAA-3', wherein the slashed part is the guard base of the cleavage site and the underlined parts are the Nde I and Xho I cleavage sites, respectively.

50. Mu.L PCR amplification System: 5×Fastpfu Buffer (Beijing full gold Biotechnology Co., ltd.) 10. Mu.L, dNTP mix (2.5 mM) 4. Mu.L, primers PfiF (10. Mu.M) and PfiR (10. Mu.M) 1. Mu.L each,FastPfu DNAPolymerase (2.5U/. Mu.L) 1. Mu.L, sterilized ultrapure water was added to 50. Mu.L, and PCR amplification was performed.

The amplification conditions were: 95 ℃ for 30sec;95 ℃,20sec, 55 ℃,30sec, 72 ℃,90sec,30 cycles; 72℃for 2min.

After the reaction, the 2.5kb target gene fragment is purified and recovered, the target gene fragment is digested by restriction enzymes Nde I and Xho I, and is connected with an expression vector pET-15b, T4 DNA ligase, and the connection product is transformed into competent cells of escherichia coli DH5 a. After single colony is picked for amplification culture, recombinant vector DNA is extracted, restriction enzymes Nde I and Xho I are used for double enzyme digestion and identification, and whether Pfu DNA polymerase mutant genes are inserted into the vector pET-15b is verified. The verification result is shown in FIG. 1, and the correct recombinant colony is cultured to obtain the recombinant expression vector containing Pfu DNA polymerase mutant.

3. Construction of Pfu DNA polymerase mutant Gene engineering Strain

The recombinant expression vector containing Pfu DNA polymerase mutant is transformed into a competent cell BL21 (DE 3) strain of escherichia coli, the strain is coated on an LB solid plate containing ampicillin with a final concentration of 100 mug/mL for overnight culture at 37 ℃, then a single strain is selected to 5mL of LB liquid medium for overnight culture at 37 ℃ and 180rpm, glycerol with a final concentration of 15% is added to obtain a Pfu DNA polymerase mutant genetic engineering strain, and the strain is packaged and stored in an ultralow temperature refrigerator at-70 ℃.

Example 2

Expression and purification of Pfu DNA polymerase mutant

1. Expression of Pfu DNA polymerase mutant

1) The Pfu DNA polymerase mutant gene engineering frozen strain obtained in example 1 was inoculated into 50mL of LB liquid medium containing 100. Mu.g/mL ampicillin, and placed in a shaking table at 37℃for shake culture overnight to obtain a seed solution.

2) Inoculating overnight cultured seed solution into 100mL LB liquid medium containing 100 μg/mL ampicillin at volume ratio of 1:100, and shaking continuously to OD in shaking table at 37deg.C ₆₀₀ About 0.6, IPTG with a final concentration of 0.5mM was added and the shaking induction was continued for 3h at 37 ℃.

3) The cells were centrifuged at 8000rpm at 4℃in a refrigerated centrifuge, and the induced cells were collected and weighed, and the wet weight of the cells was recorded.

4) After centrifugation, 150. Mu.L of lysis buffer (50 mM Tris-HCl,150mM NaCl,1mM PMSF,1%NP40,pH8.0) was added to each of the bacterial solutions before and after induction, the mixture was sonicated (power: 250W, sonicated for 5s at 15s intervals for 40 cycles), the supernatant was collected by centrifugation, 150. Mu.L of 2 XSDS loading buffer (100 mM Tris-HCl,200mM DTT,4%SDS,0.2% bromophenol blue, 20% glycerol, pH 6.8) was added to the supernatant, the mixture was boiled in boiling water for 5 minutes, and the supernatant was collected by centrifugation and analyzed by SDS-PAGE to observe the protein expression, and the results are shown in lanes 1 and 2 of FIG. 2.

2. Purification of Pfu DNA polymerase mutant

1) Taking the frozen induced expression thalli at the temperature of minus 20 ℃, adding 10mL buffer A (50mM Phosphate buffer,300mM NaCl,10mM imidazole, pH 7.0) into each gram of thalli to resuspend the thalli, and using an ultrasonic cytoclasis instrument to lyse the thalli (power 250W, ultrasonic for 5s, interval 15s, and lasting 45 cycles). Until the bacterial suspension is not sticky and clear. Then water bath at 80 ℃ for 30min. The supernatant was collected by centrifugation at 12000rpm at 4℃for 15 min. Passing through 0.45 μm filter membrane to prevent clogging of the column. 10. Mu.L of the supernatant of the bacterial sample was subjected to SDS-PAGE electrophoresis, and the result is shown in lane 3 of FIG. 1.

2) 5mL of Ni-NTA 6FF chromatographic column (Shanghai) is connected into a column position valve of a fast AKTA protein purification instrument, a buffer A cleaning system and a balancing column are firstly used, and then the filtrate obtained in the step 1 is loaded by a sample pump. After loading, the column was washed with buffer B (50mM Phosphate buffer,300mM NaCl,50mM imidazole, pH 7.0) to remove the impurity protein, and finally the target protein was eluted with elution buffer C (50mM Phosphate buffer,300mM NaCl,350mM imidazole, pH 7.0) and the elution peak was collected. 10. Mu.L of the eluted peak sample was subjected to SDS-PAGE electrophoresis, and the result is shown in lane 4 of FIG. 2.

3) Pfu DNA polymerase mutant samples collected by Ni-NTA affinity chromatography in step 2 were dialyzed overnight at 4℃in buffer D (50mM Phosphate buffer,50mM NaCl,pH7.0). And replacing the buffer C contained in the sample with the buffer D. The 5mL DEAE 6FF chromatographic column (Shanghai) was inserted into the AKTA protein purifier column position valve, the system and equilibrated column were washed with buffer D (50mM Phosphate buffer pH7.0, 50mM NaCl) and the overnight dialyzed sample was loaded with a sample pump. After loading, the column was washed with buffer D, the flow through peak was collected, and 10. Mu.L of the flow through peak sample was subjected to SDS-PAGE electrophoresis, and the results are shown in lane 5 of FIG. 2.

Example 3

Pfu DNA polymerase mutant 3'-5' exonuclease Activity and 5'-3' polymerase Activity assay

1. Pfu DNA polymerase mutant 5'-3' polymerase Activity assay

The protein concentrations of the purified Pfu DNA polymerase mutant and the wild-type Pfu DNA polymerase were first measured using a Qubit 3.0 nucleic acid protein quantitative analyzer, and then the activity measurement was performed after diluting the sample protein concentration to 10 ng/. Mu.L with a storage buffer (50 mM Tris-HCl,0.1mM EDTA,1mM DTT,0.1%Tween 20,0.1%NP40, 50% glycerol, pH 8.2). The 5'-3' polymerase activity assay system was formulated as follows: 10 Xreaction buffer (20 mM Tris-HCl,2mM MgCl) ₂ ，6mM(NH ₄ ) ₂ SO ₄ 10mM KCl,0.1%Triton X-100,0.01% BSA, pH 8.0) 5. Mu.L, 2. Mu.g/. Mu.L of activated salmon sperm DNA 5. Mu.L, 1.5mM dNTP (250 cpm/pmol [ inclusive ] ³ H]dTTP) 5. Mu.L, 10 ng/. Mu.L Pfu DNA polymerase mutant or wild-type Pfu DNA polymerase 5. Mu. L, ddH ₂ O30. Mu.L. The mixture was reacted at 74℃for 30 minutes and then placed on ice to terminate the reaction. mu.L of the reaction product was applied to DE81 ion exchange filter paper, and after spotting dried, the mixture was washed with 2 XSSC buffer (300mM NaCl,30mM Na) ₃ C ₆ H ₅ O ₇ Ph 7.0), and finally washing with ice-bath absolute ethanol once and airing, and measuring the activity of the infiltrated radioactive substance by using a liquid scintillation counter.

2. Pfu DNA polymerase mutant 3'-5' exonuclease Activity assay

The 3'-5' exonuclease activity detection system was formulated as follows: 10 Xreaction buffer (20 mM Tris-HCl,2mM MgCl) ₂ ，6mM(NH ₄ ) ₂ SO ₄ ，10mM KCl，0.1％Triton X-100，0.01％BSA，pH8.0)5μL、5μg/μL[ ³ H]Labeling of E.coli DNA 1. Mu.L, 10 ng/. Mu.L Pfu DNA polymerase mutant or wild-type Pfu DNA polymerase 5. Mu. L, ddH ₂ O39. Mu.L. The mixture was reacted at 75℃for 10 minutes and placed in ice-cooling to terminate the reaction. Subsequently, after 50. Mu.L of 0.1% BSA was added as a carrier to the reaction mixture, 100. Mu.L of a 10% TCA solution was added and mixed. The mixture was left on ice for 15 minutes and then centrifuged at 12000rpm for 10 minutes, and the supernatant was separated. Radioactivity in 100 μl of the supernatant was measured with a liquid scintillation counter to determine 3'-5' exonuclease activity.

3. Detection result

The detection results of Pfu DNA polymerase mutant and wild-type Pfu DNA polymerase 5'-3' polymerase activity and 3'-5' exonuclease activity are shown in Table 1.

TABLE 1 Pfu DNA polymerase and wild-type Pfu DNA polymerase 5'-3' polymerase Activity and 3'-5' exonuclease Activity

As can be seen from Table 1, pfu DNA polymerase mutants and wild-type Pfu DNA polymerase were detected as 32 and 61253 in terms of 5'-3' polymerase activity and as 1480 and 860 in terms of 3'-5' exonuclease activity, respectively. As shown in FIG. 3, the H147K and G387P double mutants of the wild-type Pfu DNA polymerase, i.e., the Pfu DNA polymerase mutant, not only greatly improved the 3'-5' exonuclease activity but also significantly attenuated the 5'-3' polymerase activity compared to the wild-type Pfu DNA polymerase, wherein the Pfu DNA polymerase mutant improved the 3'-5' exonuclease activity by 172% and the 5'-3' polymerase activity was only 0.05% of the wild-type Pfu DNA polymerase.

Example 4

Influence of Pfu DNA polymerase mutant on DNA polymerase family A fidelity

To determine whether Pfu DNA polymerase mutant as broad-spectrum fidelity factor will have positive feedback effect on the fidelity of DNA polymerase in DNA polymerase family A-i.e. to maximally improve the fidelity without significantly affecting the amount of PCR amplified product.

According to the invention, taq and Tth DNA polymerase in the DNA polymerase family A are selected as representatives, a ratio of adding 10-100 ng Pfu DNA polymerase mutant according to 2.5U is adopted, an improved LacI method (Cline et al, 1996) is adopted, and changes of mutation rate of the Pfu DNA polymerase mutant during amplification of LacZ alpha gene by the DNA polymerase before and after adding are reflected in a form of blue-white spot ratio, so that influence of the Pfu DNA polymerase mutant on the fidelity of Taq and Tth DNA polymerase is analyzed.

1. Influence of Pfu DNA polymerase mutant on Taq DNA polymerase fidelity

1) pUC19 plasmid DNA (containing Amp resistance gene and LacZ. Alpha. Gene) was linearized using Sca I endonuclease, purified and recovered, and diluted to 1 ng/. Mu.L plasmid DNA;

2) The entire length of pUC19 plasmid was PCR amplified using a linearized pUC19 plasmid DNA containing LacZ alpha gene as a template and Taq DNA polymerase (Thermo Co.) to which Pfu DNA polymerase mutant was not added and added, and the amplification primer sequences were as follows: pUC19-MluF:5' -AAAAACGCGTCACCAGTCACAGAAAAGCATCTTAC-3'、pUC19-MluR：5'-AAAAACGCGTCAACCAAGTCATTCTGAGAATAGT-3', wherein the slashed part is the guard base of the cleavage site and the underlined part is the Mlu I cleavage site. Specific Pfu DNA polymerase mutant addition amounts of 50. Mu.L PCR amplification system were as follows:

TABLE 2 PCR amplification System

The amplification conditions were: 95 ℃ for 2min;95 ℃,20sec, 55 ℃,20sec, 72 ℃,2min30sec,25 cycles; 72℃for 3min.

3) After the reaction, the concentration of the PCR amplified product of Taq DNA polymerase without and with Pfu DNA polymerase mutant in step 2 was detected by using a Qubit 3.0 nucleic acid protein quantitative analyzer, and the amount of amplified product was counted and calculated according to 2 ^d Template replication times d were calculated for =pcr yield/starting template amount company, and specific results are shown in table 3.

TABLE 3 Taq DNA polymerase amplification product amount and template replication times

DNA polymerase	Initial template quantity (ng)	Amplification product amount (ng)	Template replication times (d)
				Taq	2	3600	10.81
Taq+20ng Pfu Mut	2	4128	11.01
				Taq+40ng Pfu Mut	2	4685	11.19
Taq+60ng Pfu Mut	2	3960	10.95
				Taq+80ng Pfu Mut	2	2926	10.51
Taq+100ng Pfu Mut	2	2214	10.11

As is clear from Table 3, the addition of 20 to 60ng of Pfu DNA polymerase mutant Taq DNA polymerase resulted in a different degree of increase in the PCR amplification product amount compared to the non-added Taq DNA polymerase due to the correction effect of the Pfu DNA polymerase mutant, while the addition of 80 to 100ng of Pfu DNA polymerase mutant Taq DNA polymerase resulted in a significant decrease in the PCR amplification product amount compared to the non-added Taq DNA polymerase. Therefore, the effect of Pfu DNA polymerase mutant on the fidelity of Taq DNA polymerase was analyzed by selecting an addition amount of 20 to 60ng without affecting the amount of the PCR amplification product of Taq DNA polymerase.

4) The full-length pUC19 fragment of 2.7kb, which was amplified without adding 20 to 60ng Pfu DNA polymerase mutant with Taq DNA polymerase, was digested with restriction enzyme Mlu I, and ligated with T4 DNA ligase, and the ligation product was transformed into E.coli DH5a competent cells, plated on LB plates containing Amp (100 mg/ml), X-gal (8 mg/ml) and IPTG (8 mg/ml), and after overnight incubation at 37 degrees, the number of blue-white spots was counted, wherein white and light blue colonies were regarded as mutant colonies. The method for calculating the mismatch rate of the DNA polymerase comprises the following steps: mismatch rate (error rate, ER) =mf/(bp×d), where bp is the amplified laczα gene length of 324; mutation rate mf = mutant colony number/total colony number; template replication number d:2 ^d PCR yield/starting template amount.

5) The entire pUC19 fragment was amplified using the entire plasmid pUC19 (containing LacZ. Alpha. Gene) as a template, and mutation rate was calculated by blue-white spot count, reflecting the mismatch rate generated during PCR amplification of Taq DNA polymerase without and with Pfu DNA polymerase mutant added, and the results are shown in Table 4 below:

TABLE 4 mismatch and fidelity of Taq DNA polymerase without and with Pfu DNA polymerase mutant added

As can be seen from Table 4 and FIG. 4, taq DNA polymerase without Pfu DNA polymerase mutant produced 8553 blue spots, 1125 white spots, and synapsesThe transformation ratio was 0.1162, and the mismatch ratio was 3.31X10 ^-5 The method comprises the steps of carrying out a first treatment on the surface of the In contrast, taq DNA polymerase with the addition of 20ng Pfu DNA polymerase mutant produced 9785 bluish spots, 201 white spots, a mutation rate of 0.0201, and a mismatch rate of 5.17X10 ^-6 The fidelity is about 6 times of that of Taq DNA polymerase; taq DNA polymerase with the further addition of 40ng Pfu DNA polymerase mutant produced 10251 bluish plaques, 91 bluish plaques, a mutation rate of 0.0088 and a mismatch rate of 2.81×10 ^-6 The fidelity is about 11 times of that of Taq DNA polymerase; finally 60ng Pfu DNA polymerase mutant Taq DNA polymerase was added to generate 8954 bluish spots, 63 white spots, mutation rate of 0.0070, mismatch rate of 1.81×10 ^-6 The fidelity is about 18 times that of Taq DNA polymerase. In general, the mutation rate and the mismatch rate of Taq DNA polymerase added with Pfu DNA polymerase mutant are obviously reduced, and the fidelity of Taq DNA polymerase is improved by about 6-18 times.

2. Effect of Pfu DNA polymerase mutant on Tth DNA polymerase fidelity

1) pUC19 plasmid DNA (containing Amp resistance gene and LacZ. Alpha. Gene) was linearized using ScaI endonuclease, purified and recovered and diluted to 1 ng/. Mu.L plasmid DNA;

2) The entire length of pUC19 plasmid was PCR amplified using linearized pUC19 plasmid DNA containing LacZ alpha gene as a template and Tth DNA polymerase (Takara Co.) to which Pfu DNA polymerase mutant was not added and added, and the amplification primer sequences were as follows: pUC19-MluF:5' -AAAAACGCGTCACCAGTCACAGAAAAGCATCTTAC-3'、pUC19-MluR：5'-AAAAACGCGTCAACCAAGTCATTCTGAGAATAGT-3', wherein the slashed part is the guard base of the cleavage site and the underlined part is the MluI cleavage site. Specific Pfu DNA polymerase mutant addition amounts of 50. Mu.L PCR amplification system were as follows:

TABLE 5 PCR amplification System

The amplification conditions were: 95 ℃ for 2min;95 ℃,20sec, 55 ℃,20sec, 72 ℃,2min30sec,25 cycles; 72℃for 3min.

3) Reverse-rotationAfter the completion of the reaction, the concentration of the PCR amplification product of Tth DNA polymerase without and with Pfu DNA polymerase mutant in step 2 was measured by using a Qubit 3.0 nucleic acid protein quantitative analyzer, and the amount of the amplification product was counted and calculated according to 2 ^d Template replication times d were calculated for =pcr yield/starting template amount company, and specific results are shown in table 6.

TABLE 6 Tth DNA polymerase amplification product amount and template replication times

As is clear from Table 6, the addition of 20 to 60ng of the Tth DNA polymerase of the Pfu DNA polymerase mutant showed a different degree of increase in the PCR amplification product amount compared to the non-added Tth DNA polymerase due to the correction effect of the Pfu DNA polymerase mutant, whereas the addition of 80 to 100ng of the Tth DNA polymerase of the Pfu DNA polymerase mutant showed a significant decrease in the PCR amplification product amount compared to the non-added Tth DNA polymerase. Therefore, the effect of Pfu DNA polymerase mutant on the fidelity of Tth DNA polymerase was analyzed by selecting an addition amount of 20 to 60ng without affecting the PCR amplification product amount of Tth DNA polymerase.

4) The full-length fragment of pUC19 of 2.7kb, amplified by Tth DNA polymerase without 20-60 ng Pfu DNA polymerase mutant added thereto, was recovered by purification, the full-length fragment of pUC19 was digested with restriction enzyme Mlu I, T4 DNA ligase was ligated, and the ligation product was transformed into E.coli DH5a competent cells, which were plated on LB plates containing Amp (100 mg/ml), X-gal (8 mg/ml) and IPTG (8 mg/ml), and after overnight culture at 37 degrees, the number of blue-white spots was counted, wherein white and light blue colonies were regarded as mutant colonies. The method for calculating the mismatch rate of the DNA polymerase comprises the following steps: mismatch rate (error rate, ER) =mf/(bp×d), where bp is the amplified laczα gene length of 324; mutation rate mf = mutant colony number/total colony number; template replication number d:2 ^d =pcr yield/starting templateAmount of the components.

5) The entire pUC19 fragment was amplified using the entire plasmid pUC19 (containing LacZ. Alpha. Gene) as a template, and the mutation rate was calculated by blue-white spot count, reflecting the mismatch rate generated during PCR amplification of Tth DNA polymerase without and with Pfu DNA polymerase mutant, and the results are shown in Table 7:

TABLE 7 mismatch and fidelity of Tth DNA polymerase without and with Pfu DNA polymerase mutant

As can be seen from Table 7 and FIG. 5, tth DNA polymerase without Pfu DNA polymerase mutant produced 9558 bluish spots, 1210 white spots, mutation rate of 0.1124, mismatch rate of 3.22X10 ^-5 The method comprises the steps of carrying out a first treatment on the surface of the Whereas Tth DNA polymerase with the addition of 20ng Pfu DNA polymerase mutant produced 9321 bluish spots, 255 white spots, a mutation rate of 0.0266, and a mismatch rate of 5.17X10 ^-6 The fidelity is about 6 times that of Tth DNA polymerase; a further addition of 40ng of Pfu DNA polymerase mutant Tth DNA polymerase produced 8954 bluish spots, 97 white spots, a mutation rate of 0.0107 and a mismatch rate of 2.98X10 ^-6 The fidelity is about 10 times of that of Tth DNA polymerase; finally, adding 60ng Pfu DNA polymerase mutant Tth DNA polymerase produced 8564 blue spots, 63 white spots, mutation rate of 0.0073, mismatch rate of 2.10X10 ^-6 The fidelity was about 15 times that of Tth DNA polymerase. In general, the mutation rate and the mismatch rate of Tth DNA polymerase added with Pfu DNA polymerase mutant are obviously reduced, and the fidelity of the Tth DNA polymerase is improved by about 4-15 times.

Example 5

Influence of Pfu DNA polymerase mutant on DNA polymerase family B fidelity

To clarify whether Pfu DNA polymerase mutant is used as broad-spectrum fidelity factor, positive feedback effect can be generated on fidelity of DNA polymerase in DNA polymerase family B, namely, fidelity is improved to the maximum extent on the premise of not obviously influencing PCR amplified product quantity.

According to the invention, pfu, vent and Kod DNA polymerase in the DNA polymerase family B are selected as representatives, the Pfu DNA polymerase mutant is added according to the proportion of 5-25 ng Pfu DNA polymerase, an improved LacI method (Cline et al, 1996) is adopted, the change of mutation rate when the DNA polymerase amplifies LacZ alpha gene before and after the Pfu DNA polymerase mutant is added is reflected in the form of blue and white spot proportion, and the influence of the Pfu DNA polymerase mutant on the fidelity of Pfu, vent and Kod DNA polymerase is analyzed.

1. Influence of Pfu DNA polymerase mutant on Pfu DNA polymerase fidelity

1) pUC19 plasmid DNA (containing Amp resistance gene and LacZ. Alpha. Gene) was linearized using ScaI endonuclease, purified and recovered and diluted to 1 ng/. Mu.L plasmid DNA;

2) The entire length of pUC19 plasmid was PCR amplified using a linearized pUC19 plasmid DNA containing the LacZ alpha gene as a template, using Pfu DNA polymerase (Promega Co.) without and with Pfu DNA polymerase mutant, with the following amplification primer sequences: pUC19-MluF:5' -AAAAACGCGTCACCAGTCACAGAAAAGCATCTTAC-3'、pUC19-MluR：5'-AAAAACGCGTCAACCAAGTCATTCTGAGAATAGT-3', wherein the slashed part is the guard base of the cleavage site and the underlined part is the MluI cleavage site. Specific Pfu DNA polymerase mutant addition amounts of 50. Mu.L PCR amplification system were as follows:

TABLE 8 PCR amplification System

The amplification conditions were: 95 ℃ for 2min;95 ℃,20sec, 55 ℃,20sec, 72 ℃,4min,25 cycles; 72℃for 3min.

3) After the reaction was completed, pfu DNA polymerase PCR amplification product concentrations of Pfu DNA polymerase mutants not added and added in step 2 were detected using a Qubit 3.0 nucleic acid protein quantitative analyzer, the amplification product amounts were counted, and the amplification product amounts were calculated according to 2 ^d Calculation of template complex by PCR yield/initial template quantity companyThe number of times d was obtained, and the specific results are shown in Table 3.

TABLE 9 Pfu DNA polymerase amplification product amount and template replication times

DNA polymerase	Initial template quantity (ng)	Amplification product amount (ng)	Template replication times (d)
				Pfu	2	2520	10.30
Pfu+5ng Pfu Mut	2	2610	10.35
				Pfu+10ng Pfu Mut	2	2560	10.32
Pfu+15ng Pfu Mut	2	2450	10.25
				Pfu+20ng Pfu Mut	2	2020	9.98
Pfu+25ng Pfu Mut	2	1760	9.78

As is clear from Table 9, the addition of Pfu DNA polymerase of 5 to 15ng of Pfu DNA polymerase mutant showed no significant decrease in the amount of PCR amplified product as compared with the Pfu DNA polymerase not added, while the addition of Pfu DNA polymerase of 20 to 25ng of Pfu DNA polymerase mutant showed a significant decrease in the amount of PCR amplified product as compared with the Pfu DNA polymerase not added. Therefore, the effect of Pfu DNA polymerase mutant on Pfu DNA polymerase fidelity was analyzed by selecting an addition amount of 5 to 15ng without affecting the amount of Pfu DNA polymerase PCR amplified product.

4) The 2.7kb pUC19 full-length fragment amplified without adding Pfu DNA polymerase with 5-15 ng Pfu DNA polymerase mutant was recovered by purification, the pUC19 full-length fragment was digested with restriction enzyme Mlu I, T4 DNA ligase was ligated, the ligation product was transformed into E.coli DH5a competent cells, plated on LB plate containing Amp (100 mg/ml), X-gal (8 mg/ml) and IPTG (8 mg/ml), and after 37 degrees of culture overnight, the number of blue-white spots was counted, wherein white and light blue colonies were regarded as mutant colonies. The method for calculating the mismatch rate of the DNA polymerase comprises the following steps: mismatch rate (error rate, ER) =mf/(bp×d), where bp is the amplified laczα gene length of 324; mutation rate mf = mutant colony number/total colony number; template replication number d:2 ^d PCR yield/starting template amount.

5) The entire pUC19 fragment was amplified using the entire plasmid pUC19 (containing LacZ. Alpha. Gene) as a template, and mutation rate was calculated by blue-white spot count, reflecting the mismatch rate generated during PCR amplification of Pfu DNA polymerase without and with Pfu DNA polymerase mutant added, and the results are shown in Table 10 below:

table 10 mismatch and fidelity of Pfu DNA polymerase without and with Pfu DNA polymerase mutant

As can be seen from Table 10 and FIG. 6, pfu DNA polymerase without Pfu DNA polymerase mutant gave 9728 bluish spots, 72 white spots, a mutation rate of 0.00735 and a mismatch rate of 2.20X10 ^-6 The method comprises the steps of carrying out a first treatment on the surface of the Whereas Pfu DNA polymerase added with 5ng of Pfu DNA polymerase mutant produced 10311 bluish spots, 30 white spots, a mutation rate of 0.00290 and a mismatch rate of 0.86×10 ^-6 The fidelity is about 2 times that of Pfu DNA polymerase; further addition of Pfu DNA polymerase of 10ng Pfu DNA polymerase mutant resulted in 10338 bluish spots, 17 white spots, a mutation rate of 0.00134 and a mismatch rate of 0.49X10 ^-6 The fidelity is about 4 times that of Pfu DNA polymerase; finally, pfu DNA polymerase added with 15ng Pfu DNA polymerase mutant generates 10112 bluish spots, 12 white spots, mutation rate of 0.00119 and mismatch rate of 0.36×10 ^-6 The fidelity was about 6 times that of Pfu DNA polymerase. In general, the mutation rate and the mismatch rate of Pfu DNA polymerase added with the Pfu DNA polymerase mutant are obviously reduced, and the fidelity of the Pfu DNA polymerase is improved by about 2-6 times.

2. Effect of Pfu DNA polymerase mutant on the fidelity of Vent DNA polymerase

1) pUC19 plasmid DNA (containing Amp resistance gene and LacZ. Alpha. Gene) was linearized using ScaI endonuclease, purified and recovered and diluted to 1 ng/. Mu.L plasmid DNA;

2) The entire length of pUC19 plasmid was amplified by PCR using linearized pUC19 plasmid DNA containing LacZ alpha gene as a template and Vent DNA polymerase (NEB Co.) without and with Pfu DNA polymerase mutant, and the amplification primer sequences were as follows: pUC19-MluF:5' -AAAAACGCGTCACCAGTCACAGAAAAGCATCTTAC-3'、pUC19-MluR：5'-AAAAACGCGTCAACCAAGTCATTCTGAGAATAGT-3', wherein the slashed part is the guard base of the cleavage site and the underlined part is the MluI cleavage site. Specific Pfu DNA polymerase mutant addition amounts of 50. Mu.L PCR amplification system were as follows:

TABLE 11 PCR amplification System

The amplification conditions were: 95 ℃ for 2min;95 ℃,20sec, 55 ℃,20sec, 72 ℃,4min,25 cycles; 72℃for 3min.

3) After the reaction was completed, the concentration of the Vent DNA polymerase PCR amplified product without and with Pfu DNA polymerase mutant in step 2 was detected using a Qubit 3.0 nucleic acid protein quantitative analyzer, the amount of amplified product was counted, and the amount was calculated according to 2 ^d Template replication times d were calculated for =pcr yield/starting template amount company, and specific results are shown in table 3.

TABLE 12 Vent DNA polymerase amplification product amount and template replication times

As is clear from Table 12, the Vent DNA polymerase added with 5 to 15ng Pfu DNA polymerase mutant showed no significant decrease in the amount of PCR amplified product as compared with the Vent DNA polymerase not added, while the Vent DNA polymerase added with 20 to 25ng Pfu DNA polymerase mutant showed significant decrease in the amount of PCR amplified product as compared with the Vent DNA polymerase not added. Therefore, the effect of Pfu DNA polymerase mutant on Vent DNA polymerase fidelity was analyzed by selecting an addition amount of 5 to 15ng without affecting the amount of Pfu DNA polymerase PCR amplified product.

4) Purifying and recovering 2.7kb pUC19 full-length fragment amplified by Vent DNA polymerase without adding Pfu DNA polymerase mutant 5-15 ng, cutting pUC19 full-length fragment by restriction enzyme Mlu I, connecting by T4 DNA ligase, transferring the connecting product into E.coli DH5a competent cell, coating on a DNA containing Amp (10)0 mg/ml), X-gal (8 mg/ml) and IPTG (8 mg/ml), and after overnight incubation at 37 degrees, the number of bluish-white spots was counted, wherein white and bluish-blue colonies were regarded as mutant colonies. The method for calculating the mismatch rate of the DNA polymerase comprises the following steps: mismatch rate (ER) =mf/(bp×d), where bp is the amplified laczα gene length of 324; mutation rate mf = mutant colony number/total colony number; template replication number d:2 ^d PCR yield/starting template amount.

5) The entire pUC19 fragment was amplified using the entire plasmid pUC19 (containing LacZ. Alpha. Gene) as a template, and mutation rate was calculated by blue-white spot count, reflecting the mismatch rate generated during PCR amplification of Vent DNA polymerase without and with Pfu DNA polymerase mutant, and the results are shown in Table 10 below:

TABLE 13 Vent DNA polymerase mismatch Rate and fidelity without and with Pfu DNA polymerase mutant

As can be seen from Table 13 and FIG. 7, vent DNA polymerase without Pfu DNA polymerase mutant produced 9899 bluish spots, 106 white spots, a mutation rate of 0.01059, and a mismatch rate of 3.11X10 ^-6 The method comprises the steps of carrying out a first treatment on the surface of the On the other hand, vent DNA polymerase added with 5ng Pfu DNA polymerase mutant produced 9867 bluish spots, 45 white spots, a mutation rate of 0.00454, and a mismatch rate of 1.33X10 ^-6 The fidelity is about 2 times that of Vent DNA polymerase; vent DNA polymerase with the further addition of 10ng Pfu DNA polymerase mutant produced 9841 bluish spots, 27 white spots, mutation rate of 0.00274, mismatch rate of 0.81×10 ^-6 The fidelity is about 3 times that of Vent DNA polymerase; finally, vent DNA polymerase added with 15ng Pfu DNA polymerase mutant produced 8994 bluish spots, 17 white spots, mutation rate of 0.00189 and mismatch rate of 0.56X10 ^-6 The fidelity was about 5 times that of Vent DNA polymerase. In general, mutation rate and mismatch rate of Vent DNA polymerase added with Pfu DNA polymerase mutant are obviously reduced, and fidelity of Vent DNA polymerase is improved by about 2-5 times.

3. Effect of Pfu DNA polymerase mutant on KOD DNA polymerase fidelity

1) pUC19 plasmid DNA (containing Amp resistance gene and LacZ. Alpha. Gene) was linearized using ScaI endonuclease, purified and recovered and diluted to 1 ng/. Mu.L plasmid DNA;

2) The entire length of pUC19 plasmid was PCR amplified using LacZ. Alpha. Gene-containing linearized pUC19 plasmid DNA as a template and KOD DNA polymerase (Toyobo Co.) without and with Pfu DNA polymerase mutant, and the amplification primer sequences were as follows: pUC19-MluF:5' -AAAAACGCGTCACCAGTCACAGAAAAGCATCTTAC-3'、pUC19-MluR：5'-AAAAACGCGTCAACCAAGTCATTCTGAGAATAGT-3', wherein the slashed part is the guard base of the cleavage site and the underlined part is the MluI cleavage site. Specific Pfu DNA polymerase mutant addition amounts of 50. Mu.L PCR amplification system were as follows:

TABLE 14 PCR amplification System

The amplification conditions were: 95 ℃ for 2min;95 ℃,20sec, 55 ℃,20sec, 72 ℃,2min30sec,25 cycles; 72℃for 3min.

3) After the reaction was completed, the concentration of the KOD DNA polymerase PCR amplified product without and with Pfu DNA polymerase mutant in step 2 was detected using a Qubit 3.0 nucleic acid protein quantitative analyzer, the amount of amplified product was counted, and the amplification product was determined according to 2 ^d Template replication times d were calculated for =pcr yield/starting template amount company, and specific results are shown in table 3.

TABLE 15 KOD DNA polymerase amplification product amount and template replication times

DNA polymerase	Initial template quantity (ng)	Amplification product amount (ng)	Template replication times (d)
				KOD	2	3520	10.78
KOD+5ng Pfu Mut	2	3663	10.84
				KOD+10ng Pfu Mut	2	3785	10.89
KOD+15ng Pfu Mut	2	3482	10.77
				KOD+20ng Pfu Mut	2	3128	10.61
KOD+25ng Pfu Mut	2	2845	10.47

As is clear from Table 15, the addition of 5 to 15ng of Pfu DNA polymerase mutant KOD DNA polymerase did not significantly reduce the amount of PCR amplified product compared to the amount of KOD DNA polymerase not added, whereas the addition of 20 to 25ng of Pfu DNA polymerase mutant KOD DNA polymerase significantly reduced the amount of PCR amplified product compared to the amount of KOD DNA polymerase not added. Therefore, the effect of Pfu DNA polymerase mutant on KOD DNA polymerase fidelity was analyzed by selecting an addition amount of 5 to 15ng without affecting the amount of Pfu DNA polymerase PCR amplified product.

4) The pUC19 full-length fragment of 2.7kb amplified by KOD DNA polymerase without 5-15 ng Pfu DNA polymerase mutant added thereto was recovered by purification, the pUC19 full-length fragment was digested with restriction enzyme Mlu I, T4 DNA ligase was ligated, and the ligation product was transformed into E.coli DH5a competent cells, which were plated on LB plates containing Amp (100 mg/ml), X-gal (8 mg/ml) and IPTG (8 mg/ml), and after culturing overnight at 37 degrees, the number of blue-white spots was counted, wherein white and light blue colonies were regarded as mutant colonies. The method for calculating the mismatch rate of the DNA polymerase comprises the following steps: mismatch rate (error rate, ER) =mf/(bp×d), where bp is the amplified laczα gene length of 324; mutation rate mf = mutant colony number/total colony number; template replication number d:2 ^d PCR yield/starting template amount.

5) The entire pUC19 fragment was amplified using the entire plasmid pUC19 (containing LacZ. Alpha. Gene) as a template, and the mutation rate was calculated by blue-white spot count, reflecting the mismatch rate generated during PCR amplification of KOD DNA polymerase without and with Pfu DNA polymerase mutant, and the results are shown in Table 10 below:

table 16 mismatch and fidelity of KOD DNA polymerase without and with Pfu DNA polymerase mutant added

As is clear from Table 16 and FIG. 8, KOD DNA polymerase without Pfu DNA polymerase mutant produced 8789 bluish spots, 141 white spots, a mutation rate of 0.01579, and a mismatch rate of 4.52X10 ^-6 The method comprises the steps of carrying out a first treatment on the surface of the Whereas KOD DNA polymerase with 5ng Pfu DNA polymerase mutant produced 8938 bluish spots, 67 white spots, mutation rate of 0.00774, mismatch rate2.12×10 ^-6 The fidelity is about 2 times that of KOD DNA polymerase; KOD DNA polymerase with the further addition of 10ng Pfu DNA polymerase mutant produced blue spots 9516, 40 white spots, mutation rate of 0.00419, mismatch rate of 1.19X10 ^-6 The fidelity is about 3 times that of KOD DNA polymerase; finally, KOD DNA polymerase with 15ng Pfu DNA polymerase mutant produced blue spots 9079, white spots 26, mutation rate 0.00286, mismatch rate 0.82×10 ^-6 The fidelity was about 5 times that of KOD DNA polymerase. In general, the mutation rate and the mismatch rate of KOD DNA polymerase added with Pfu DNA polymerase mutant are both obviously reduced, and the fidelity of KOD DNA polymerase is improved by about 2-5 times.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. A mutant of a high-fidelity Pfu DNA polymerase, wherein the amino acid sequence of the mutant comprises a mutation in the 3'-5' exonuclease activity domain and the 5'-3' polymerase domain of the high-fidelity Pfu DNA polymerase such that the exonuclease activity in the 3'-5' exonuclease activity domain is increased and the polymerase activity in the 5'-3' polymerase domain is decreased.

2. The mutant of claim 1, wherein the amino acid sequence of the mutant comprises a mutation in the 3'-5' exonuclease active domain of a high-fidelity Pfu DNA polymerase: histidine to lysine, mutation occurs in the 5'-3' polymerase domain: glycine is mutated to proline.

3. The mutant according to claim 1 or 2, wherein when the amino acid sequence of the high-fidelity Pfu DNA polymerase is SEQ ID No.2, the following mutation occurs in the amino acid sequence of the mutant: H147K and G387P.

4. A mutant according to claim 3, wherein the amino acid sequence of the mutant is as shown in SEQ ID No. 1.

5. A mutant gene according to any one of claims 1 to 4.

6. A recombinant vector comprising the coding gene of claim 5.

7. A recombinant host expressing the mutant of any one of claims 1-4.

8. The recombinant host of claim 7, wherein the system for expressing the mutant in the recombinant host comprises a prokaryotic expression system.

9. A method for preparing the mutant according to any one of claims 1 to 4 using a prokaryotic expression system, comprising the steps of: ligating the coding gene of claim 5 into a prokaryotic expression vector to obtain a recombinant vector;

transforming bacteria by using the recombinant vector to obtain recombinant bacteria;

inducing the recombinant bacteria to express, wherein the expression product contains the mutant.

10. Use of a mutant according to any one of claims 1 to 4 to improve the fidelity of a DNA polymerase.