WO2014205882A1 - High-fidelity dna polymerase, and preparation and use thereof - Google Patents

Abstract

Provided are a high-fidelity DNA polymerase, a gene for coding the high-fidelity DNA polymerase, a method for preparing the high-fidelity DNA polymerase, and a use thereof in nucleic acid amplification. The high-fidelity DNA polymerase has an amino acid sequence represented by SEQ ID NO:1, can be used to amplify a nucleic acid template with a high GC content or a nucleic acid template with a low sample purity, and can amplify a very low template amount.

Description

 High-fidelity DNA polymerase and preparation and application thereof

 This application claims priority to Chinese Patent Application No. 201310268966.9, entitled "A High-Fidelity DNA Polymerase and Its Preparation and Application", filed on June 28, 2013, the entire contents of which is hereby incorporated by reference. Combined in this application. Technical field

 The invention belongs to the field of genetic engineering and relates to a high fidelity DNA polymerase and its preparation and application. Background technique

 DNA polymerases are widely used in molecular biology research, such as DNA sequencing, labeling, mutation, and nucleic acid amplification. Heat-resistant DNA polymerase is not attenuated under the conditions of high temperature denaturation of nucleic acid due to its thermal stability, and is a commonly used enzyme for nucleic acid amplification. Depending on the sequence, DNA polymerase can be divided into six major groups: A, B, C, D, X and Y. The heat-resistant DNA polymerase mainly belongs to group A and group B, and has similar biological properties. Representative of the A-group DNA polymerase is Taq DNA polymerase, which is isolated from the genome of The s aquaticus YT-1, having 5,-3, exonuclease activity and 5,-3, DNA polymerase activity. Its strain is grown in hot springs around 75 °C. Because it can withstand high temperature denaturation, it has been widely used in PCR since its discovery. It has been developed into a variety of kits; later studies have been isolated from other species. A variety of Group A DNA polymerases have emerged, but because they do not have the exonuclease-correcting activity of 3,-5, it is easy to incorporate the wrong base during amplification, resulting in the inability to faithfully amplify the template until the B group. The discovery of DNA polymerases made it possible to amplify templates with high fidelity. The B-group DNA polymerase is mainly isolated from archaea, including the archaea, the archaea, the cyanobacteria and the archaea, which have higher thermal stability, and the 3,-5, exonuclease-correcting activity can be recognized. The bases in the extended strand are incorrectly incorporated, and they are excised, and the DNA polymerase activity is added as a base complementary to the template, thereby ensuring high consistency between the newly amplified strand and the template.

Hyperthermophilic archaea has strict anaerobic and hyperthermophilic growth characteristics, such as PyrocHciium and ^awc^ym? species can grow at 10 ° C, but below 80 ° C can not grow; from Daxi >Ofob^/M an7 isolated from the 3650 m superheated chimney of the mid-ocean ridge is the most heat-resistant archaea known at present, which can be increased at high temperatures up to 113 ° C, and stopped below 90 ° C. Growing. Many DNA polymerases have been isolated from the OCOCO« and 73⁄4er ococo« genus. Some DNA polymerases remain stable at very high temperatures and are used in molecular biology research, especially in nucleic acid amplification. Among them, Pfo DNA polymerase is a thermostable DNA polymerase derived from ^racoco^/Mn'om?, having 5,-3, DNA polymerase activity and exonuclease activity of 3,-5. Since Pfu DNA polymerase has exonuclease activity of 3,-5, the probability of error in nucleic acid amplification is greatly reduced, and the fidelity is about 6 times that of Taq DNA polymerase. At present, Pfo DNA polymerase is usually used as the preferred high-fidelity DNA polymerase for high-fidelity amplification of nucleic acids. However, in practical applications, Pfo DNA polymerase requires high structure, purity and concentration of primers and samples. The reaction sensitivity is low and the amplification efficiency is low, which limits the use and promotion of Pfu DNA polymerase. Summary of the invention

 In view of the above, an object of the present invention is to provide a high-sensitivity, high-amplification high-fidelity DNA polymerase, and its preparation and use.

 In order to achieve the object of the present invention, the present invention uses the following technical solutions:

 The present invention provides a high-fidelity DNA polymerase, designated Tco DNA polymerase, having any one of the amino acid sequences shown in (1), (II):

 (I) having the amino acid sequence of SEQ ID NO: 1;

 (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

 The invention provides a Tco DNA polymerase for use in nucleic acid amplification, and the Tco DNA polymerase has any one of the amino acid sequences shown by (I) and (II):

 (I) having the amino acid sequence of SEQ ID NO: 1;

 (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

The present invention provides a DNA molecule encoding Tco DNA polymerase, which has I, Any one of the nucleotide sequences shown in II:

 I has the nucleotide sequence shown in SEQ ID NO: 2;

 a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown by SEQ ID NO: 2;

 TcoDNA polymerase has any one of the amino acid sequences shown in (1) and (II):

(I) having the amino acid sequence shown by SEQ ID NO:1;

 (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

 The present invention provides a recombinant vector comprising a DNA molecule encoding a Tco DNA polymerase having any one of the nucleotide sequences represented by I and II: I having the core represented by SEQ ID NO: Glycosidic acid sequence;

 a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown by SEQ ID NO: 2;

 The Tco DNA polymerase has any one of the amino acid sequences shown in (I) and (II): (I) having the amino acid sequence of SEQ ID NO: 1;

 (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

 Preferably, the above recombinant vector is a recombinant vector containing a T7 promoter.

 The present invention provides a PCR amplification kit comprising Tco DNA polymerase having any one of the amino acid sequences shown in (1) and (II):

 (I) having the amino acid sequence shown by SEQ ID NO:1;

 (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

 The present invention also provides a method for preparing a high-fidelity DNA polymerase, comprising the steps of: obtaining a DNA molecule having an amino acid sequence encoding as defined in (I) or (II); and merging the above DNA molecule with an expression vector to construct a recombinant Expression vector;

 Transferring the above recombinant expression vector into a host cell to obtain a transformant;

Inducing the above transformant to express the fusion protein, and separating and purifying to obtain high fidelity DNA polymerization Enzyme

 (I) is: having the amino acid sequence of SEQ ID NO: 1;

 (Π) is: an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

 Preferably, the DNA molecule having an amino acid sequence as defined in (I) or (II), specifically having any one of the nucleotide sequences shown by I, II:

 I has the nucleotide sequence shown in SEQ ID NO: 2;

 II A nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown by SEQ ID NO: 2.

 Preferably, a DNA molecule having an amino acid sequence encoding as defined in (I) or (II) is obtained, specifically, the above DNA molecule is recombinantly constructed from the hyperthermophilic archaea Thermococcus coalescens; the above hyperthermophilic ancient The fungus was purchased from the Japan Collection of Microorganisms and Cultures, and its deposit number is JCM: 12540. It is isolated from the ultra-high temperature seawater of the Shuiyuanhai Mountain of the Izu Ogasawara, and it is an irregular spherical shape of 0.5-2 μΜ. It can be fused during the exponential growth phase. It is a fusion cell of about 5 μΜ; the bacteria is grown at 57-90 ° C, pH 5.2-8.7, its optimum pH is 6.5, and the optimal growth temperature is 87 ° C, which has super thermophilic growth characteristics;

 (I) is: having the amino acid sequence of SEQ ID NO: 1;

 (Π) is: an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of the amino acid sequence shown by SEQ ID NO: 1.

 Preferably, the host cell used in the preparation method of the high fidelity DNA polymerase is a prokaryotic system host cell.

 Preferably, the host cell used in the preparation method of the high fidelity DNA polymerase is an E. coli host cell.

 Preferably, the purification operation in a method for preparing a high-fidelity DNA polymerase, specifically, purification using a gel medium.

 In some embodiments of the invention, a method of making Tco DNA polymerase is specifically:

(1) Nucleotide sequence of different DNA polymerase genes by the homologous ratio of Thermococcus 歹'J, designing primers with higher similarity segments, amplifying the Thermococcus coalescens DNA polymerase gene fragment, and then continuously amplifying the full-length sequence of the gene according to the obtained sequence information as shown in SEQ ID NO: Nucleotide sequence;

 (2) analyzing the above gene structure, amplifying the mature protein coding sequence, and obtaining a gene fragment as shown in SEQ ID NO: 2 by overlapping PCR, and constructing the vector into the vector containing the T7 promoter to obtain a recombinant vector;

 (3) transforming the above recombinant vector into a host cell E. coli Rosetta (DE3) to obtain a transformant capable of producing the high-fidelity DNA polymerase;

 (4) The transformant is expanded and used to prepare the high-fidelity DNA polymerase after induction; (5) the high-fidelity DNA polymerase is obtained by sequentially purifying using a nickel gel medium and a heparin chromatography column.

 The present invention provides a high fidelity DNA polymerase, a preparation method thereof and use in nucleic acid amplification. The high-fidelity DNA polymerase has the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1. The experimental data show that the purity of the high-fidelity DNA polymerase prepared by the preparation method disclosed in the present invention is over 95%, and the high-fidelity DNA polymerase provided by the present invention can be expanded compared with the Pfu high-fidelity DNA polymerase. Increased GC content of nucleic acid template, nucleic acid template with low sample purity, and also can achieve very low template amount amplification, greatly improving the sensitivity and amplification efficiency of high-fidelity DNA polymerase, while having a higher The fidelity is suitable for all kinds of PCR and experiments with high requirements for amplification fidelity, and is also suitable as a high-fidelity DNA polymerase in a PCR amplification kit. DRAWINGS

 Figure 1 shows the results of purification of Tco DNA polymerase; wherein Lane 1 shows purified Tco DNA polymerase; Lane M shows protein molecular weight Marker;

Figure 2 shows the results of Tco DNA polymerase amplifying nucleic acid fragments of different lengths; wherein, Lane M shows DNA molecular weight Marker; Lane 1 shows human genome 800 bp β-Actin fragment; Lane 2 shows human genome 1.3 kb β-globin fragment; Lane 3 shows Genomic 1.5kb β-globin tablets Segment; lane 4 shows the human genomic 3.0 kb β-globin fragment; lane 5 shows the 4.1 kb β-globin fragment; lane 6 shows the λ genome 8 kb fragment;

 Figure 3 shows the results of Tco DNA polymerase and Pfo DNA polymerase amplifying high GC content nucleic acid template and inhibitor template; among them, 1 ~ 4 shows Tco DNA polymerase amplification results; a ~ d shows Pfu DNA polymerase expansion Increased results; Lane M shows DNA molecular weight Marker; Lane 1 shows 237 bp fragment of human whole blood; Lane 2 shows 1.5 kb 16 srDNA fragment of E. coli; Lane 3 shows 2 kb fragment of Thermus thermophilus HB8 gene; Lane 4 shows 1.5 kb fragment of human genome; Lane a A full-blood 237 bp fragment is shown; lane b shows a 1.5 kb 16 sr DNA fragment of E. coli; lane c shows a 2 kb fragment of Thermus thermophilus HB8 gene; and lane d shows a human genomic 1.5 kb fragment;

 Figure 4 shows the results of amplification of different template amounts of pUC19 plasmid by Tco DNA polymerase and Pfu DNA polymerase; 1 to 5 shows Tco DNA polymerase amplification results; 6 to 10 shows Pfli DNA polymerase amplification results; lane M DNA molecular marker Marker; Lane 1 shows 10 ng of pUC19; Yongdao 2 shows lng of pUC19; Yongdao 3 shows 0.1 ng of pUC19; Yongdao 4 shows O.Olng of pUC19; Lane 5 shows O.OOlng of pUC19 Lane 6 shows 10 ng of pUC19; lane Ί shows lng of pUC19; Lane 8 shows O. lng of pUC19; Lane 9 shows O.Olng of pUC19; Lane 10 shows O.OOlng of pUC19;

Figure 5 shows the results of Tco DNA polymerase and Pfu DNA polymerase amplifying different templating amounts of human genomic DNA; wherein 1-4 shows Tco DNA polymerase amplification results; 5-8 shows Pfu DNA polymerase amplification results; M shows DNA molecular weight Marker; Lane 1 shows 25 ng of the human genome 1.3 kb fragment; Lane 2 shows 10 ng of the human genome 1.3 kb fragment; Lane 3 shows 5 ng of the human genome 1.3 kb fragment; Lane 4 shows 1 ng of the human genome 1.3 kb fragment; Lane 5 shows 25 ng of the human genome 1.3 kb fragment; Lane 6 shows 10 ng of the human genome 1.3 kb fragment; Lane 7 shows 5 ng of the human genome 1.3 kb fragment; Lane 8 shows 1 ng of the human genome 1.3 kb fragment; Figure 6 shows different DNA aggregation Enzyme amplification error rate; wherein 1 shows Taq DNA polymerase; 2 shows Pfu DNA polymerase; 3 shows Tco DNA polymerase. detailed description The invention discloses a high-fidelity DNA polymerase, Tco DNA polymerase, and a preparation method of the high-fidelity DNA polymerase and application thereof in nucleic acid amplification. Those skilled in the art can refer to the content herein to obtain the high-fidelity DNA polymerase, and realize its application. It should be particularly noted that all similar substitutions and modifications will be apparent to those skilled in the art, and they are all regarded as It is included in the present invention. The preparation method and application of the present invention have been described in the preferred embodiments, and it is obvious that those skilled in the art can make modifications or appropriate changes and combinations to the preparation methods and applications herein without departing from the scope of the invention. The technique of the present invention is applied.

 The high fidelity DNA polymerase provided by the present invention and the reagents and materials used in the preparation and use thereof are commercially available.

 In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further illustrated by the following examples: Example 1 Acquisition of DNA molecules encoding Tco DNA polymerase

 The species Thermococcus coalescens, purchased from the Sakamoto Biological Collection, is JCM: 12540. The purchased strain was centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded. The pellet was precipitated and the genomic DNA was extracted with reference to Promega's Wizard® Genomic DNA Purification kit. The brief steps are as follows:

 The cell pellet was resuspended in 600 μΙ^ Nuclei Lysis Solution, and the cells were lysed in a water bath at 80 ° C for 5 min. After cooling to room temperature, add 20 (^L Protein Preciptitation Solution, shake and mix, ice bath for 5 min; centrifuge at 12000 rpm for 5 min; absorb the supernatant to another A clean centrifuge tube, add 600 μΙ isopropyl alcohol, mix and freeze in ice bath for 10 min; centrifuge at 12000 rpm for 5 min; nucleic acid precipitation is washed twice with 700 μ∑ 70% ethanol, centrifuged at 12000 rpm for 5 min, the supernatant is discarded, and the nucleic acid precipitate is dried at room temperature for 10 min. To the taste of no alcohol, add 50 μΙ^ ΤΕ to dissolve, and obtain the genomic DNA of the strain.

The obtained Thermococcus coalescens genomic DNA was not subjected to whole genome sequencing, and thus the gene sequence of its DNA polymerase could not be obtained. By BLAST similarity analysis, the regions with high similarity of DNA polymerase sequences of the same sequence were obtained, and the primers were designed to obtain the Tco DNA polymerase coding sequence. Search the sequenced archaeal DNA polymerase from the NCBI database The long gene and its upstream and downstream lkb nucleotide sequences were designed using the Clustal W software to compare the Thermococcus gammatolerans EJ3, Pyrococcus yayanosii CHI, Thermococcus kodakarensis KOD1, Thermococcus onnurineus NA1, Thermococcus sibiricus MM 739 sequence 歹¹ J , and designed primers:

 Primer BamHITcoF: having the nucleotide sequence as shown in SEQ ID NO: 5;

 Primer Tco493R: having the nucleotide sequence as shown in SEQ ID NO: 6;

 Primer Tco3085F: having the nucleotide sequence as shown in SEQ ID NO: 7; primer TcoDownR: having the nucleotide sequence as shown in SEQ ID NO: 8;

Using the extracted genomic DNA of the strain as a template, the PCR reaction solution was prepared according to the following system: 5 (^L system contains: 50 ng of the above genomic DNA, 400 nmol/L of primers, dNTPs 20 (^mol/L, 5 x Prime STAR® Buffer ( Mg ²⁺ plus ) 1 ΟμΙ^ , BioScience's PrimeSTAR®HS 0.5μΙ^ PCR amplification procedure: 98 °C 2min, (94 °C 10s; 55 °C 20s; 68 °C 3min) 25 cycles, extension at 68 °C for 5 min. PCR product electrophoresis detection, using primer BamHITcoF+Tco493R to amplify a fragment of about 2.5 kb, primer Tco3085F+TcoDownR amplification to obtain a fragment of about 3 kb, respectively recovering the target band, and using Taq separately. After DNA polymerase was added to A, the obtained two DNA molecules were respectively ligated into the pGM-T vector, and the positive clone was confirmed by sequencing.

 The nucleotide sequence obtained by the above sequencing was confirmed by NCBI to be the most similar to the DNA polymerase B gene of Thermococcus and Pyrococcus, and the similarity with Thermococcus sp. AM4 DNA polymerase B was 79%. What is obtained is the coding sequence of Tco DNA polymerase. Analysis of the sequence revealed that the encoded DNA polymerase contained an intein, and the two amplified sequences could not be spliced into a full-length gene.

 Design primers based on sequencing results and analysis results:

 Primer Tco30F: having the nucleotide sequence as shown in SEQ ID NO: 9;

Using Tco30F and TcoDownR as primers, the extracted genomic DNA was used as a template, and a fragment of about 3.5 kb was amplified and constructed into a pGM-T vector, which was sequenced. The sequence was also found to contain intein. According to the sequencing result, the 3.5 kb fragment was spliced with a fragment of about 2.5 kb amplified by the primer BamHITcoF+Tco493R to obtain Tco DNA polymerase. The full-length gene has a full sequence comprising 6186 bp, the sequence of which is shown in SEQ ID NO: 3, and the encoded protein is 2061 aa, and the amino acid sequence thereof is shown in SEQ ID NO: 4.

 Comparing the spliced gene sequence with the coding sequence of other DNA polymerase mature proteins in the NCBI database, the Tco DNA polymerase gene was divided into four coding frames by three inteins, and the gene including 2328 bp was removed after the intein was removed. The encoded mature protein is 775 aa.

 According to the Tco DNA polymerase mature protein coding sequence obtained by the analysis, primers were designed to amplify four ORFs:

 Primer Tcol2R: having the nucleotide sequence as shown in SEQ ID NO: 10;

 Primer TcoORF12F: having the nucleotide sequence shown as SEQ ID NO: 1 1; primer NTcoORF2R: having the nucleotide sequence shown as SEQ ID NO: 12; primer NTcoORF23F: having the sequence shown in SEQ ID NO: 13. Nucleotide sequence; primer TcoORF3R: having the nucleotide sequence shown as SEQ ID NO: 14;

 Primer NTcoORF34F: having the nucleotide sequence shown as SEQ ID NO: 15; primer NotlTcoWR: having the nucleotide sequence shown as SEQ ID NO: 16; using the extracted genomic DNA as a template, PrimeSTAR amplification In different coding frames, BamfflTcoF and Tcol2R were amplified to obtain about 1.2 kb of ORF1, TcoORF12F and NTcoORF2R were amplified to obtain about 250 bp of ORF2, NTcoORF23F and TcoORF3R were amplified to obtain about 150 bp of ORF3, NTcoORF34F and NotlTcoWR were amplified to obtain about 700 bp of ORF4. The four ORFs were ligated to the pGM-T vector, sequenced and aligned with the spliced full-length gene, and the results showed - corresponding.

 Add the same molar amount of ORF1, 2, 3, 4 fragments, and perform overlapping PCR with the primers BamfflTcoF and NotlTcoWR, and splicing to obtain the full-length coding region, whose sequence is shown in SEQ ID NO: 2, and the amino acid of the encoded protein. The sequence is as shown in SEQ ID NO: 1, and the resulting full-length about 2.3 kb fragment is recovered, and the resulting fragment is a DNA molecule encoding Tco DNA polymerase. Example 2 Construction of Recombinant Vector Containing DNA Molecules Encoding Tco DNA Polymerase

The target fragment of about 2.3 kb in length obtained in Example 1 was digested with BamH I and Not I, ligated into the same digested T7 promoter-containing vector, and transformed into DH5a competent form. In the cell.

 Through the PCR identification, restriction enzyme digestion and sequencing verification, the correctness of the gene sequence encoding Tco DNA polymerase was confirmed, the correct competent cells were cultured and the plasmid was extracted, and the obtained plasmid was encoded with Tco DNA polymerase. Recombinant vector for DNA molecules. Example 3 Preparation of transformants expressing Tco DNA polymerase fusion protein

The recombinant vector containing the DNA molecule encoding Tco DNA polymerase was obtained in Example 2 and transformed into a host cell. Co/Rosetta (DE3), single colony culture was picked until OD _6QQ = 0.4, and IPTG was added to a concentration of 0.1 mmol. After induced by /L for 4 h, the cells were sonicated and the expression of the target protein was detected by SDS-PAGE. The obtained transformants were able to express Tco DNA polymerase fusion protein. Example 4 Preparation of Tco DNA Polymerase

The transformant strain capable of expressing the Tco DNA polymerase fusion protein obtained in Example 3 was inoculated to a 500 mL Erlenmeyer flask containing 200 mL of LB medium at a ratio of 1:100, and kanamycin was added thereto to a final concentration of 30 mg/ L and chloramphenicol were brought to a final concentration of 34 mg/L, and cultured at 37 ° C at 200 rpm to OD ₆ . . =0.4, IPTG was added to its final concentration of 0.1 mmol/L, and induced at 30 °C for 4 h; the induced bacterial solution was collected, centrifuged at 12,000 rpm for 2 min, the supernatant was discarded, and the cells were pH 7.8 with 20 mL/20 mmol/L Tris- HCl, O.lmol/L KCl, 50mmol/L NaCl buffer A plus 30mmol/L imidazole resuspended, frozen in -80°C refrigerator for 12h; 37°C lysed cells, ultrasonically disrupted at 70°C water bath After treatment for 30 min, centrifuge at 12000 rpm for 10 min; the supernatant was purchased from the National Biochemical Engineering Technology Research Center and eluted with buffer A containing 400 mmol/L imidazole; the eluate was purchased from the National Biochemical Engineering Technology Research Center. The affinity medium was eluted with buffer A containing 200 mmol/L, 400 mmol/L, 600 mmol/L, and 1 mol/L NaCl; each protein peak was collected and detected by SDS-PAGE, and buffer A containing 200 mmol/L NaCl was found. A part of the target protein can be eluted, buffer A containing 400 mmol/L NaCl is eluted with a large amount of the target protein, and the obtained product is dialyzed to Storage buffer: pH 7.4, 20 mmol/L Tris-HCl, 0.1 mmol/L EDTA, 0.1% Tween 20, 0.5% Nonidet P40, Ol mol/L KCl, 50% glycerol.

 The results showed that Tco DNA polymerase was successfully obtained. SDS-PAGE showed the purification results as shown in Figure 1: Lane 1 shows purified Tco DNA polymerase; Lane M shows the molecular weight of the marker, Marker. As can be seen from the figure, the obtained Tco DNA polymerase has a molecular weight of about 90 kDa, which is consistent with its theoretical size, and its purity is more than 95% by BandScan software. Example 5 Determination of Tco DNA Polymerase Activity

 The Tco DNA polymerase prepared in Example 4 was assayed for its activity at a nucleotide concentration of 30 min at 72 ° C, diluted to 2.5 uL, and tested for its 3,-5, exonuclease activity by fluorescent probe method. The results showed that Tco DNA polymerase had strong corrective activity, indicating that its amplification fidelity performance was better. In order to test the thermostability of Tco DNA polymerase, the enzyme was incubated at 90 ° C, and the DNA polymerase activity was measured at 0, 1, 2, 3, 4, 5, 6 h. The results showed that Tco DNA polymerase had a pole. High thermal stability with a 90°C half-life of 6 h. Example 6 Application of Tco DNA Polymerase in Nucleic Acid Amplification

The Tco DNA polymerase prepared in Example 4 was applied to nucleic acid amplification. First, the reaction conditions of the Tco DNA polymerase in nucleic acid amplification were tested. Using pUC19 plasmid as a template, primers were designed to amplify about 2.7 kb fragment to determine the optimum pH value of the reaction and K ⁺ , NH ₄ ⁺ , Mg ^{2 . +} ion concentration. Prepare 10 X buffer B: 500mmol/L Tris-HCl, pH 7.4~8.8, 15mmol/L MgCl ₂ , add different concentrations of KC1, (NH ₄ ) ₂ S0 ₄ , the reaction system is 20μΙ^, which contains: different Conditions 10 X buffer B 2 L, 2.5 mmol/L dNTPs 1.6 μΙ^, ΙΟμηιοΙ/L primer PAs 0^ , ΙΟμηιοΙ/L primer PAa 0.5μΙ, Tco DNA polymerase 0.2 L, 15ng^L pUC19 0.5 μΙ^, according to 98 °C 2min, (98 °C 10s, 60 °C 20s, 68 °C 2min40s) 25cycles, the last 68 °C extension 5min, electrophoresis detection, determine the optimal buffer. According to the results, the optimal reaction conditions for determining Tco DNA polymerase were 10 X Tco buffer: pH 8.4, 500 mmol/L Tris-HCl, 150 mmol/L KC1, 100 mmol/L (NH ₄ ) ₂ S0 ₄ , 15 mmol/L MgCl ₂ .

The 10 x Tco buffer and the Tco DNA polymerase prepared in Example 4 were respectively Amplification: Human genomic DNA of about 800 bp P-Actin, 1.3 kb, 1.5 kb, 3.0 kb, and 4.1 kb β-globin fragments and λ genome of 8 kb fragment, agarose gel electrophoresis results showed that they were well amplified. Target segment. As a result of agarose gel electrophoresis, the results are shown in Figure 2, wherein the lanes indicate DNA molecular weight Marker; Lane 1 shows the human genome 800 bp β-Actin fragment; Lane 2 shows the human genome 1.3 kb β-globin fragment; Lane 3 shows the human genome 1.5 kb Β-globin fragment; lane 4 shows the human genomic 3.0 kb β-globin fragment; lane 5 shows the 4.1 kb β-globin fragment; lane 6 shows the λ genome 8 kb fragment. Example 7 Tco DNA Polymerase for High GC Content Nucleic Acid Templates and Amplified Thermophilic Microorganisms Containing Inhibitor Templates In order to adapt to high temperature environments, the genome contains higher GC content, such as

The Thermus thermophilus HB8 genome has a GC content of about 70%. It is difficult to amplify such a template for ordinary PCR reactions. However, some templates are susceptible to contamination by inhibitors in the material source during the purification process, resulting in failure of the PCR reaction, such as Anticoagulant EDTA, heparin, etc. in the blood.

In order to test the amplification effect of the Tco DNA polymerase prepared in Example 4 on such complex template and inhibitor-containing template, a 237 bp SOX21 non-coding fragment was directly PCR amplified using human whole blood containing EDTA as a template; Approximately 1.5 kb fragment of 16srDNA was amplified by Bacillus lysate as a template; the high-GC fragment of about 2 kb was amplified with the Thermus thermophilus HB8 genome as a template, and its GC content was about 72%. The human genome was used as a template to amplify it to a height of about 1.5 kb. The GC fragment has a GC content of about 65.9%. The Pfo DNA polymerase purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. is used as a comparative solution, and is amplified according to the optimum conditions. After the reaction is completed, the same amount of product is electrophoresed. . Tco DNA polymerase can be well amplified to obtain the desired band, while the comparative example has no target band. As a result of agarose gel electrophoresis, the results are shown in Figure 3. Among them, 1 ~ 4 shows the results of Tco DNA polymerase amplification; a ~ d shows the results of Pfo DNA polymerase amplification; Lane M shows the molecular weight of Marker; 237 bp fragment of whole blood; Lane 2 shows 1.5 kb 16 srDNA fragment of Escherichia coli; Lane 3 shows 2 kb fragment of Thermus thermophilus HB8 gene; Lane 4 shows 1.5 kb fragment of human genome; Lane a shows 237 bp fragment of human whole blood; Lane b shows Escherichia coli 1.5 kb 16 srDNA fragment of bacterial solution; Lane 2 shows a 2 kb fragment of Thermus thermophilus HB8 gene; Lane d shows a human genomic 1.5 kb fragment. Example 8 Amplification ability of Tco DNA polymerase under different template amounts

 Using the pUC19 plasmid as a template, the 2.7 kb pUC19 plasmid was amplified using the Tco DNA polymerase prepared in Example 4. 10 ng, lng, 0.1 ng, O.Olng, 0.00 lng UC19 plasmid, 5 (^L reaction system, Tco DNA polymerase prepared in Example 4 and Pfu purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. DNA polymerase was amplified according to the optimal conditions. After the reaction was completed, 5μ product electrophoresis was detected. The results of agarose gel electrophoresis showed that the amplification sensitivity of Tco DNA polymerase was much higher than that of Pfu DNA polymerase. 1 to 5 shows Tco DNA polymerase amplification results; 6-10 shows Pfli DNA polymerase amplification results; Lane M shows DNA molecular weight Marker; Lane 1 shows 10 ng of pUC19; Lane 2 shows lng of pUC19; Lane 3 O. lng pUC19; Lane 4 shows O.Ongng pUC19; Lane 5 shows O.OOlng of pUC19; Lane 6 shows 10 ng of pUC19; Lane 7 shows lng of pUC19; Lane 8 shows O. lng of pUC19; Lane 9 O.Olng pUC19; Lane 10 shows O.OOlng pUC19. It can be observed from Figure 4 that when 50μΙ^ reaction system is used to amplify the plasmid template, Tco DNA polymerase can detect O.Olng pUC19 plasmid, while Pfu DNA polymerase Amplify this only under 10 ng conditions The tablets. Example 9 different amounts of template ^ ^, Tco DNA polymerase amplification activity

Using the human genome as a template, the human 1.3 kb fragment was amplified using the Tco DNA polymerase prepared in Example 4. 25g, 10ng, 5ng, lng of human genomic DNA, 50μΙ reaction system, Tco DNA polymerase prepared in Example 4 and Pfu DNA polymerase purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Conditional amplification, after completion of the reaction, 5 L product electrophoresis was detected. Agarose gel electrophoresis results showed that the amplification sensitivity of Tco DNA polymerase was much higher than that of Pfo DNA polymerase. The electrophoresis results are shown in Figure 5, where 1-4 shows Tco DNA polymerase amplification results; 5-8 shows Pfli DNA polymerase amplification results; Lane M shows DNA molecular weight Marker; Lane 1 shows 25 ng human genome 1.3 kb Fragment 2 shows 10 ng of the human genome 1.3 kb fragment; Lane 3 shows 5 ng of the human genome 1.3 kb fragment; Lane 4 shows 1 ng of the human genome 1.3 kb fragment; Lane 5 shows 25 ng of the human genome 1.3 kb fragment; Lane 6 shows a 10 ng human genome 1.3 kb fragment; lanes indicate 5 ng of the human genome 1.3 kb fragment; Lane 8 shows a lng human genome 1.3 kb fragment. From Figure 5, Tco DNA polymerase can amplify as little as 5 ng of human genomic template, while Pfu DNA polymerase can only obtain weak amplification at a maximum template amount of 25 ng. Example 10 Tco DNA polymerase fidelity test

The fidelity of the Tco DNA polymerase prepared in Example 4 was tested by the method of blue-white spot, and the full-length plasmid was amplified by using TUC DNA polymerase, Taq DNA polymerase and Pfu DNA polymerase, respectively, using the pUC19 plasmid as a template. Amplification factor of each enzyme. The amplification factor d is in accordance with the formula: 2 ^d = amount of PCR product / amount of starting template; Z3⁄4w I digested the template plasmid and digested, ligated and transformed DH5a cells, and coated a plate containing IPTG and the chromogenic substrate X-Gal. And count blue and white spots. The mutation frequency mf, in accordance with the formula mf = white spot number / total number of colonies. The error rate of different DNA polymerase amplification was calculated by the formula ER=mf/(bpxd). The fidelity of DNA polymerase was 1/ER, and the lower the error rate, the better the fidelity. The results showed that Tco DNA polymerase had good fidelity performance, and its fidelity was 1.8 times that of Pfu and 9.2 times that of Taq. The experimental results, Figure 6 shows the error rate of the three, where 1 shows Taq DNA polymerase; 2 shows Pfli DNA polymerase; 3 shows Tco DNA polymerase. The error rate of Tco DNA polymerase is: 1.2 χ 1θΛ The error rate of Pfo DNA polymerase is: 2.2 10" ⁶ , the error rate of Taq DNA polymerase is: 10.9 10" ⁶ „ According to the fidelity of DNA polymerase is 1/ ER: The fidelity of Tco DNA polymerase is 1.8 times that of Pfu DNA polymerase and 9.2 times that of Taq DNA polymerase. From the above examples, a high-fidelity DNA polymerase disclosed in the present invention, namely Tco DNA, is known. Polymerase, which has the ability to amplify different templates and fragments, and also has good results for complex templates and inhibitor-containing templates, and has high fidelity amplification ability, suitable for all kinds of PCR and requirements for amplification fidelity. High experiments are also suitable as high-fidelity DNA polymerases in PCR amplification kits.

The above is only a preferred embodiment of the present invention, and it should be noted that the above-described preferred embodiments should not be construed as limiting the scope of the invention. The square is subject to accuracy. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and scope of the invention.

Claims

Rights request 1. A high-fidelity DNA polymerase, characterized in that it has any one of the amino acid sequences shown in (1) and (II): (I) Having the amino acid sequence shown in SEQ ID NO: 1; (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1. 2. Application of the high-fidelity DNA polymerase as claimed in claim 1 in nucleic acid amplification. 3. A DNA molecule encoding the high-fidelity DNA polymerase as claimed in claim 1, characterized in that it has any one of the nucleotide sequences shown in I and II: I has the nucleotide sequence shown in SEQ ID NO:2; II has a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO:2. 4. A recombinant vector, characterized in that it contains the DNA molecule as claimed in claim 3. 5. The recombinant vector according to claim 4, characterized in that it is a recombinant vector containing a T7 promoter. 6. A PCR amplification kit, characterized in that it contains a high-fidelity DNA polymerase; the high-fidelity DNA polymerase has any one of the amino acid sequences shown in (I) and (II): (1) Having the amino acid sequence shown in SEQ ID NO: 1; (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1. 7. A method for preparing high-fidelity DNA polymerase, characterized by comprising the following steps: Obtain a DNA molecule encoding an amino acid sequence defined as (I) or (II); fuse the DNA molecule with an expression vector to construct a recombinant expression vector; Transfer the recombinant expression vector into a host cell to obtain a transformant; The transformant is induced to express the fusion protein, and after isolation and purification, a high-fidelity DNA polymerase is obtained; Described (1) is: having the amino acid sequence shown in SEQ ID NO: 1; Described (II) is: an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1. 8. The preparation method according to claim 7, characterized in that the DNA molecule has any one of the nucleotide sequences shown in I and II: I has the nucleotide sequence shown in SEQ ID NO:2; II has a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO:2. 9. The preparation method according to claim 7, characterized in that the host cell is a prokaryotic system host cell. 10. The preparation method according to claim 9, characterized in that the prokaryotic system host cell is Escherichia coli.