CN118006581A

CN118006581A - Pfu DNA polymerase mutant strain with XNA recognition and synthesis activity and application thereof

Info

Publication number: CN118006581A
Application number: CN202410164761.4A
Authority: CN
Inventors: 陈庭坚; 陶睿; 马星雲; 黎志聪; 谢玲玲; 杜宇辉
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2024-02-05
Filing date: 2024-02-05
Publication date: 2024-05-10

Abstract

The invention discloses Pfu DNA polymerase mutant strain with XNA recognition and synthesis activity and application thereof, comprising Pfu DNA polymerase mutant strain P1 and P1 exo ^‑; the Pfu DNA polymerase mutant strain can efficiently recognize and synthesize RNA and 2'-F and 2' -OMe modified nucleic acid. Compared with the wild type, the mutant strain P1 can transcribe RNA and 2' -F-DNA with high fidelity, reverse transcribe 2' -F-DNA and 2' -OMe-DNA, and synthesize RNA and 2' -F-DNA by taking the 2' -F-DNA as a template; compared with the wild type, the P1 exo ^‑ can efficiently transcribe 2'-F-DNA and reverse transcribe 2' -F-DNA and 2'-OMe-DNA under the condition of not adding Mn ²⁺, and simultaneously the activity of synthesizing the 2' -OMe-DNA by using different templates under the condition of adding 1mM Mn ²⁺ is obviously improved.

Description

Pfu DNA polymerase mutant strain with XNA recognition and synthesis activity and application thereof

Technical Field

The invention relates to the field of enzyme molecular transformation, in particular to Pfu DNA polymerase mutant strain with XNA recognition and synthesis activity and application thereof.

Background

Natural nucleic acid DNA and RNA are vectors for all vital genetic information, and are also the basis for the storage, use and transfer of genetic information in all cells. However, DNA and RNA are poorly biochemically stable, greatly limiting their use in many applications. In recent years, many DNA and RNA analogs have been developed that can be used as useful biomaterial building elements or functional molecules, with the ability to encode genetic information and support evolution. These synthetic genetic polymers are known as unnatural nucleic acids (XNA). The development of unnatural nucleic acids has mainly involved modification of the base, sugar ring and phosphate groups. Among them, the most widely studied is XNA in which sugar rings are replaced or modified. Non-natural nucleic acids in which pentoses of DNA or RNA are replaced mainly include Hexanol Nucleic Acid (HNA), cyclohexene nucleic acid (CeNA), locked Nucleic Acid (LNA), arabinose Nucleic Acid (ANA), 2' -fluoro-arabinose nucleic acid (FANA), threose Nucleic Acid (TNA), and the like. Among non-natural nucleic acids in which sugar rings are modified, the modification of the 2 'position of ribose or deoxyribose is mainly studied, because the modification of the 2' position can change the structure of the sugar ring itself, affect the conformation of the whole nucleic acid molecule, thereby affecting its binding capacity and changing the activity of the molecule. Sugar ring modifications include the substitution of hydrogen or hydroxyl groups in the sugar ring with various atoms or groups, including 2 '-fluoro (2' -F), 2 '-azide (2' -Az), 2 '-amino (2' -Am), and 2 '-methoxy (2' -OMe) modifications, and the like. Although only one atom or group in pentoses is substituted in these modifications, these modifications result in significant changes in the properties of DNA or RNA. For example, 2'-F and 2' -OMe modifications greatly increase the melting temperature, duplex stability, and nuclease resistance of nucleic acids. Thus, XNA presents great advantages in the development of nucleic acid aptamers and biomaterials with greater biostability.

DNA polymerase, RNA polymerase and reverse transcriptase are the main executors of DNA and RNA synthesis in life, play a key role in the storage, use and transfer of genetic information, and are also extremely important tools for biotechnology, disease diagnosis and protein engineering. However, natural nucleic acid polymerases are highly selective for substrates, which often prevents them from recognizing and utilizing unnatural substrates, and thus the transfer of genetic information between DNA and XNA, RNA and XNA, and different XNA, such as transcription, reverse transcription and replication of XNA, to each other is difficult to achieve. To solve this problem, researchers have designed and engineered nucleic acid polymerases by directed evolution and rational or semi-rational design methods to enable their identification, utilization and synthesis of non-natural nucleic acids.

Currently, some groups have succeeded in engineering polymerase mutants capable of recognizing and synthesizing unnatural nucleic acids. For example, the Romesberg group has obtained Stoffel fragment mutants SFM4-3, SFM4-6 and SFM4-9 of Taq DNA polymerase by directed evolution, wherein SFM4-3 can synthesize not only all 2' -OMe modified non-natural nucleic acids, but also partial 2' -OMe or 2' -F modified nucleic acids by PCR amplification. The Ellington group obtained by screening T7 RNA polymerase mutant strains capable of synthesizing 2' -OMe modified non-natural nucleic acid, and obtained mutant strains RGVG-M5 and RGVG-M6 with better heat stability. Although the ability of these polymerase mutants to recognize and utilize unnatural substrates is greatly improved after modification, they suffer from low fidelity and low heat resistance. The source strains of the B-group DNA polymerase live in an extreme environment with higher temperature, have higher heat resistance, and have verification activity naturally and higher fidelity. There have also been some attempts to engineer group B DNA polymerases, such as Holliger group evolved a series of Tgo polymerase mutants that were able to synthesize and reverse transcribe various XNAs, such as HNA, TNA, FANA and ANA, etc. The Obika group was engineered to obtain KOD mutants capable of efficiently synthesizing long-fragment LNA, 2' -OMe-modified nucleic acids. The Ellington group obtains KOD mutant strains which can efficiently reverse transcribe 2' -OMe-DNA and have verification activity through screening and evolution.

Pfu DNA polymerase is Pyrococcus furiosus from the genus Thermomyces extremophila, a member of the family B DNA polymerase, and has greater thermostability and higher fidelity of DNA synthesis than many group A DNA polymerases and some other group B DNA polymerases. Therefore, molecular engineering of Pfu DNA polymerase is expected to obtain one or more Pfu DNA polymerase mutant strains with higher fidelity and thermal stability, which can efficiently identify and synthesize XNA, and meet the urgent need of realizing wide application of XNA on excellent XNA polymerase.

Disclosure of Invention

The primary object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide Pfu DNA polymerase mutant strains capable of efficiently recognizing and synthesizing unnatural nucleic acids (XNA).

Another object of the present invention is to provide the use of the Pfu DNA polymerase mutant strain described above.

The aim of the invention is achieved by the following technical scheme:

A Pfu DNA polymerase mutant strain P1 has an amino acid sequence shown in SEQ ID NO. 1.

Further, the Pfu DNA polymerase mutant strain P1 is obtained by introducing the following mutations ：I38L、A40V、V93Q、R97M、K118I、I137L、E251K、G350V、V353L、L381R、V390I、K467R、K469N、A486L、F494L、G499A、T515I、I522L、F588L、E665R、S712V、N736K and W769R into Pfu DNA polymerase.

A Pfu DNA polymerase mutant strain P1 exo ^- has an amino acid sequence shown in SEQ ID NO. 2.

Further, the Pfu DNA polymerase mutant strain P1 exo ^- is obtained by introducing the following mutation into the Pfu DNA polymerase mutant strain P1: d141A and E143A.

A gene encoding the Pfu DNA polymerase mutant strain P1; further, the gene sequence is shown as SEQ ID NO. 3.

A gene encoding the Pfu DNA polymerase mutant strain P1 exo ^-; further, the gene sequence is shown as SEQ ID NO. 4.

An expression cassette or vector comprising the gene encoding the Pfu DNA polymerase mutant strain P1 or the gene encoding the Pfu DNA polymerase mutant strain P1 exo ^-.

A recombinant bacterium or recombinant cell comprising the gene encoding the Pfu DNA polymerase mutant strain P1, the gene encoding the Pfu DNA polymerase mutant strain P1 exo ^-, an expression cassette or a vector.

The Pfu DNA polymerase mutant strain P1, the Pfu DNA polymerase mutant strain P1 exo ^-, an expression cassette, a vector, a recombinant bacterium or a recombinant cell are applied to the synthesis of non-natural nucleic acid (XNA).

Further, the non-natural nucleic acid includes a non-natural nucleic acid modified with a sugar ring; still further, the non-natural nucleic acid is a 2'-OMe or 2' -F modified nucleic acid.

A kit for synthesizing nucleic acid, comprising Pfu DNA polymerase mutant strain P1 or Pfu DNA polymerase mutant strain P1 exo ^-.

Further, the nucleic acid comprises a non-natural nucleic acid (XNA); still further, the non-natural nucleic acid includes a non-natural nucleic acid modified with a sugar ring; still further, the non-natural nucleic acid is a 2'-OMe or 2' -F modified nucleic acid.

Further, the kit also comprises a template, a primer, a reagent containing Mn ²⁺ and a substrate. Further, the Mn ²⁺ -containing reagent is MnCl ₂.

Compared with the prior art, the invention has the following advantages and effects:

Compared with the wild type, the two Pfu DNA polymerase mutants (Pfu DNA polymerase mutant strain P1 and Pfu DNA polymerase mutant strain P1 exo ^-) provided by the invention can transcribe RNA and 2' -F-DNA, reverse transcribe 2' -F-DNA and 2' -OMe-DNA with high fidelity, and synthesize RNA and 2' -F-DNA by taking 2' -F-DNA as a template; compared with the wild type, the P1 exo ^- can efficiently transcribe 2'-F-DNA and reverse transcribe 2' -F-DNA and 2'-OMe-DNA under the condition of not adding Mn ²⁺, and simultaneously the activity of synthesizing the 2' -OMe-DNA by using different templates under the condition of adding 1mM Mn ²⁺ is obviously improved.

Drawings

FIG. 1 is a schematic representation of the structure of nucleoside triphosphates used in the synthesis of DNA, RNA, 2'-F-DNA and 2' -OMe-DNA.

FIG. 2 is a schematic diagram of Pfu DNA polymerase synthesizing DNA, RNA, 2'-F-DNA and 2' -OMe-DNA using DNA/RNA/2'-F-DNA/2' -OMe-DNA as templates.

FIG. 3 is a graph showing the results of activity test of wild-type Pfu DNA polymerase and its mutants for synthesizing RNA, 2'-F-DNA and 2' -OMe-DNA using DNA as a template; wherein A is an activity test result diagram of synthetic RNA, B is an activity test result diagram of synthetic 2'-F-DNA, and C is an activity test result diagram of synthetic 2' -OMe-DNA.

FIG. 4 is a graph showing the results of activity tests of wild-type Pfu DNA polymerase and its mutants in synthesizing DNA, RNA, 2'-F-DNA and 2' -OMe-DNA using RNA as a template; wherein A is an activity test result diagram of synthetic DNA, B is an activity test result diagram of synthetic RNA, C is an activity test result diagram of synthetic 2'-F-DNA, and D is an activity test result diagram of synthetic 2' -OMe-DNA.

FIG. 5 is a graph showing the results of activity tests of wild-type Pfu DNA polymerase and its mutants in synthesizing DNA, RNA, 2' -F-DNA and 2' -OMe-DNA using 2' -F-DNA as a template; wherein A is an activity test result diagram of synthetic DNA, B is an activity test result diagram of synthetic RNA, C is an activity test result diagram of synthetic 2'-F-DNA, and D is an activity test result diagram of synthetic 2' -OMe-DNA.

FIG. 6 is a graph showing the results of activity tests of wild-type Pfu DNA polymerase and its mutants in synthesizing DNA, RNA, 2' -F-DNA and 2' -OMe-DNA using 2' -OMe-DNA as a template; wherein A is an activity test result diagram of synthetic DNA, B is an activity test result diagram of synthetic RNA, C is an activity test result diagram of synthetic 2'-F-DNA, and D is an activity test result diagram of synthetic 2' -OMe-DNA.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1

(1) Biological material for experiments

The E.coli DH5 a/pET 28a (+) employed in this example, DH5 a competent cells, pET28a (+) plasmids were routinely obtained by commercial means; wherein the pET28a (+) plasmid can also be extracted from E.coli DH5 alpha/pET 28a (+) using a high purity plasmid miniprep kit (Tiangen Biochemical technologies (Beijing) Co., ltd.). pET28a (+) -P1 is synthesized by the Optimago company and has the sequence shown in SEQ ID NO. 5.

(2) Obtaining pET28a (+) -P1 exo ^-

The inventors designed mutations ：I38L、A40V、V93Q、R97M、K118I、I137L、E251K、G350V、V353L、L381R、V390I、K467R、K469N、A486L、F494L、G499A、T515I、I522L、F588L、E665R、S712V、N736K and W769R at the following sites for Pfu DNA polymerase Gene (Gene ID: 1468044); and synthesized pET28a (+) -P1 (synthesized by the Rheumatoid biosystems; the sequence is shown as SEQ ID NO. 5) containing Pfu DNA polymerase mutant P1 gene, wherein the P1 gene sequence is shown as SEQ ID NO. 3.

Performing overlap extension PCR to mutate the P1 gene at fixed points to obtain Pfu DNA polymerase gene (P1 exo ^- gene) containing mutation D141A, E A, wherein the mutated sequence is shown as SEQ ID No. 4.

Specifically, the primer sequences used for overlap extension PCR site-directed mutagenesis are shown in Table 1, and the specific steps are as follows:

1) Respectively amplifying the DNA fragment 1 and the DNA fragment 2 by using pET28a (+) -P1 as a template, wherein the reaction systems are shown in a table 2 and a table 3, and the PCR procedures are shown in a table 4 and a table 5; after the PCR reaction is finished, recovering the DNA fragment by using an agarose gel DNA recovery kit (Guangzhou Meiyi Biotechnology Co., ltd.) to obtain a DNA fragment 1 (with a sequence shown as SEQ ID NO. 6) and a DNA fragment 2 (with a sequence shown as SEQ ID NO. 7) for the next overlap extension PCR;

2) Overlap extension PCR, the PCR reaction system and PCR procedure used are shown in tables 6 and 7, respectively; after the PCR reaction was completed, a DNA fragment 3 (sequence shown as SEQ ID NO. 8) containing the P1 exo ^- gene and the pET28a (+) upstream and downstream homology arms was recovered using an agarose gel DNA recovery kit (Guangzhou Mei-based Biotech Co., ltd.).

3) Ligation of the P1 exo ^- Gene and plasmid pET28a (+

The DNA fragment 3 and the plasmid pET28a (+) were digested with restriction enzymes NcoI and XhoI, and placed in a digestion reaction system, which was incubated at 37℃for 3 hours, and the digestion reaction system is shown in Table 8. After completion of the cleavage reaction, the resulting DNA fragment 3 and the resulting plasmid pET28a (+) were recovered using an agarose gel DNA recovery kit (Guangzhou Mei-based biotechnology Co., ltd.).

The digested DNA fragment 3 and digested plasmid pET28a (+) were ligated with T4 DNA ligase, and incubated overnight at 16℃in an enzyme-linked reaction system, which is shown in Table 9, and after the enzyme-linked reaction, the obtained enzyme-linked product was purified using an agarose gel DNA recovery kit (Guangzhou Mei-based Biotech Co., ltd.) to obtain pET28a (+) -P1 exo ^-.

(3) Transforming cells and extracting plasmids

And respectively converting pET28a (+) -P1 and enzyme-linked product pET28a (+) -P1 exo ^- synthesized by the biological company into competent cells of escherichia coli clone strain DH5 alpha by an electric shock conversion method to obtain recombinant bacteria DH5 alpha/pET 28a (+) -P1 and DH5 alpha/pET 28a (+) -P1 exo ^-. Positive clones were verified by colony PCR, plasmids were extracted, and sequenced.

(4) Preparation of recombinant expression strains

Recombinant plasmids pET28a (+) -P1 and pET28a (+) -P1 exo ^- with correct sequencing are extracted from recombinant bacteria DH5 alpha/pET 28a (+) -P1 and DH5 alpha/pET 28a (+) -P1 exo ^-, and transferred into escherichia coli expression strain BL21 (DE 3) pLysS by a shock transformation method to obtain recombinant expression strains BL21 (DE 3) pLysS/pET28a (+) -P1 and BL21 (DE 3) pLysS/pET28a (+) -P1 exo ^-.

TABLE 1PCR amplification and site-directed mutagenesis primer sequences

TABLE 2 PCR reaction System for DNA fragment 1

TABLE 3 PCR reaction System for DNA fragment 2

TABLE 4 PCR reaction procedure for DNA fragment 1

TABLE 5 PCR reaction procedure for DNA fragment 2

TABLE 6 PCR reaction System for DNA fragment 3

TABLE 7 PCR reaction procedure for DNA fragment 3

TABLE 8 cleavage reaction System for DNA fragment 3 and plasmid

TABLE 9 enzymatic ligation reaction System of DNA fragment 3 and vector after cleavage

Example 2

Single colonies of BL21 (DE 3) pLysS/pET28a (+) -P1 and BL21 (DE 3) pLysS/pET28a (+) -P1 exo ^- were picked up and inoculated into 20mL of 2 XYT medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL chloramphenicol, respectively, and cultured overnight at 37℃and 220 rpm. The overnight culture was transferred to 1L of 2 XYT medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL chloramphenicol, cultured at 37℃and 220rpm until the absorbance value OD ₆₀₀ = 0.6-0.8, added with 0.5mM IPTG (isopropyl thiogalactoside), and cultured at 18℃for 16-20h. After centrifugation at 6000rpm for 10min at 4℃and collection of the cells, they were resuspended in Buffer A (50 mM Tris-HCl, 150mM NaCl, 5mM imidazole (imidazole), 0.1mM EDTA, 0.1% Triton X-100, pH 7.0) and disrupted in a high pressure homogenizer for 15min at 75℃in a water bath. After centrifugation at 10000rpm at 4℃for 45min, the supernatant was subjected to removal of cell debris with a 0.45 μm filter, and the filtered supernatant was purified by nickel column affinity chromatography. After 1-2h incubation of the protein supernatant with nickel column, the protein of interest was eluted with 1 XElutation buffer (50 mM Tris-HCl, 150mM NaCl, 10-500mM imidazole, 0.1mM EDTA, 0.1% Triton X-100, pH 7.0) containing different concentrations of imidazole. The resulting product was confirmed by SDS-PAGE (polyacrylamide gel electrophoresis). The target protein was further dialyzed and concentrated using 50kDa Amicon-Ultra centrifugal ultrafiltration tube and 1 XDialysis buffer (100 mM Tris-HCl, 1mM EDTA, 2mM DTT, 0.2% Tween 20, pH 7.6), during which the Dialysis 1 XDialysis buffer was repeatedly changed multiple times. Obtaining purified Pfu DNA polymerase mutant strain P1 and Pfu DNA polymerase mutant strain P1 exo ^- protein after dialysis, finally adding 100% glycerol with the same volume for dilution and storing in a refrigerator at-20 ℃; for subsequent example studies.

Example 3

Activity test study of polymerase mutants P1, P1 exo ^- for recognition of DNA templates specifically, the activities of polymerase mutants P1, P1 exo ^- of the present invention were tested by the following experiments to synthesize RNA, 2'-F-DNA and 2' -OMe-DNA products using DNA as templates without addition of Mn ²⁺ and with addition of 1mM Mn ²⁺. The schematic structure of nucleoside triphosphates for the synthesis of DNA, RNA, 2'-F-DNA and 2' -OMe-DNA is shown in FIG. 1. A schematic diagram of Pfu DNA polymerase for synthesizing DNA, RNA, 2'-F-DNA and 2' -OMe-DNA using DNA/RNA/2'-F-DNA/2' -OMe-DNA as a template is shown in FIG. 2.

Template T58 and complementary primer FAM-P18 are mixed in a ratio of 2:1 in advance, denatured at 95 ℃ for 10min, slowly cooled to room temperature, incubated on ice for 5min, and then the rest reagents are complemented. The DNA template and the complementary primer sequences are shown in Table 10; the reaction system for synthesizing the full-length DNA product control is shown in Table 11; the reaction systems for synthesizing RNA, 2'-F-DNA and 2' -OMe-DNA using Pfu polymerase wild type, polymerase mutant P1, P1exo ^- are shown in Table 12; the reaction procedure is shown in Table 13. When rNTPs is used as a substrate, 1U/. Mu.L of RNase inhibitor is added into the reaction system. When rNTPs or 2' -F-dNTPs are used as a substrate, 1 mu M enzyme (Pfu polymerase wild type, polymerase mutant strain P1 or polymerase mutant strain P1exo ^-) is added into the reaction system; when 2' -OMe-dNTPs were used as substrates, 2. Mu.M enzyme (Pfu polymerase wild-type, polymerase mutant strain P1 or polymerase mutant strain P1exo ^-) was added to the reaction system. After the reaction was completed, twice the volume of 2 XTBE-Urea loading buffer was added to the product, denatured at 95℃for 10min, and finally the product was analyzed by 20% denatured polyacrylamide gel electrophoresis.

TABLE 10DNA templates and complementary primer sequences

TABLE 11 reaction System for Taq DNA polymerase to synthesize full-length DNA product control Using DNA as template

Table 12Pfu DNA polymerase reaction System for synthesizing different nucleic acid molecules Using DNA as template

TABLE 13 reaction procedure for synthesizing different nucleic acid molecules Using DNA as templates

The result of electrophoresis of each reaction product is shown in FIG. 3.

Lane 1 in fig. 3A is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using DNA as template without Mn ²⁺ added; lanes 6-8, from left to right, are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 3B is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesized fully modified 2' -F-DNA using DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of fully modified 2' -F-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 3C is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesized fully modified 2' -OMe-DNA using DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of fully modified 2' -OMe-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using DNA as template with the addition of 1mM Mn ²⁺.

In each figure, P is the 18nt primer band position and F is the 58nt full-length product band position of DNA. The bands above the full length band position are bands resulting from incomplete denaturation of the product. When the wild-type Pfu DNA polymerase and the mutant strain P1 cannot synthesize a product, the primer may be degraded because it does not remove the exonuclease activity.

As can be seen from FIG. 3A, under the condition that Mn ²⁺ is not added, the mutant strain P1 can synthesize the full-length product of a small amount of RNA by taking DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing RNA by taking DNA as the template of the mutant strain P1 is obviously improved; under the condition of adding 1mM Mn ²⁺, the wild Pfu DNA polymerase can synthesize a small amount of full-length products of RNA by taking DNA as a template, and the mutant strains P1 and P1 exo ^- can synthesize a large amount of full-length products of RNA by taking DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing RNA by taking DNA as a template of the mutant strains P1 and P1 exo ^- is obviously improved.

As can be seen from FIG. 3B, the wild-type Pfu DNA polymerase synthesizes a certain amount of the full-length product of 2'-F-DNA by using DNA as a template without adding Mn ²⁺, but has a partial truncated product, while P1 synthesizes a large amount of the full-length product of 2' -F-DNA by using DNA as a template, and P1 exo ^- synthesizes a certain amount of the full-length product of 2'-F-DNA by using DNA as a template and almost no truncated product, so that the activity of synthesizing 2' -F-DNA by using DNA as a template of mutant strains P1 and P1 exo ^- is remarkably improved as compared with the wild-type Pfu DNA polymerase. Under the addition of 1mM Mn ²⁺, the wild-type Pfu DNA polymerase, mutant P1 and mutant P1 exo ^- were able to synthesize the full-length product of 2' -F-DNA with almost no truncated product using DNA as template.

As can be seen from FIG. 3C, under the condition that Mn ²⁺ is not added, the mutant strains P1 and P1 exo ^- can synthesize a certain amount of truncated products of 2'-OMe-DNA by taking DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing 2' -OMe-DNA by taking DNA as a template of the mutant strains P1 and P1 exo ^- is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strain P1 can synthesize a certain amount of full-length products of 2' -OMe-DNA by taking DNA as a template, and the mutant strain P1 exo ^- can synthesize a small amount of full-length products of 2' -OMe-DNA by taking DNA as a template, so that compared with wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing the 2' -OMe-DNA by taking DNA as a template is obviously improved.

Example 4

Activity test study of polymerase mutants P1, P1 exo ^- for recognition of RNA templates specifically, the activities of polymerase mutants P1, P1 exo ^- of the present invention were tested by the following experiments to synthesize DNA, RNA, 2'-F-DNA and 2' -OMe-DNA products using RNA as templates without addition of Mn ²⁺ and with addition of 1mM Mn ²⁺.

The template T30-1 or RNA-T30 and the complementary primer FAM-P15 are mixed in a ratio of 2:1 in advance, denatured at 65 ℃ for 5min, slowly cooled to room temperature, incubated on ice for 5min, and then the rest reagents are complemented. The DNA, RNA templates and complementary primer sequences are shown in Table 14; the reaction system for the control of the full length product of the synthetic DNA is shown in Table 15; the reaction systems for Pfu polymerase wild type, polymerase mutant P1, P1 exo ^- to synthesize DNA, RNA, 2'-F-DNA and 2' -OMe-DNA are shown in Table 16; the reaction procedure is shown in Table 17. When dNTPs, rNTPs and 2' -F-dNTPs are used as substrates, 1 mu M enzyme (Pfu polymerase wild type, polymerase mutant strain P1 or polymerase mutant strain P1 exo ^-) is added into the reaction system; when 2' -OMe-dNTPs were used as substrates, 2. Mu.M enzyme (Pfu polymerase wild-type, polymerase mutant strain P1 or polymerase mutant strain P1 exo ^-) was added to the reaction system. After the reaction was completed, twice the volume of 2 XTBE-Urea loading buffer was added to the product, denatured at 95℃for 10min, and finally the product was analyzed by 20% denatured polyacrylamide gel electrophoresis.

TABLE 14DNA, RNA templates and complementary primer sequences

R: ribonucleotides

TABLE 15Taq DNA polymerase reaction System for synthesizing full-length DNA product control Using DNA as template

TABLE 16Pfu DNA polymerase reaction System for synthesizing different nucleic acid molecules Using RNA as template

TABLE 17 reaction procedure for synthesizing different nucleic acid molecules Using DNA and RNA as templates

The result of electrophoresis of each reaction product is shown in FIG. 4.

Lane 1 in fig. 4A is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of DNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using RNA as template without Mn ²⁺ added; lanes 6-8, from left to right, are the products of DNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using RNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 4B is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using RNA as template without Mn ²⁺ added; lanes 6-8, from left to right, are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using RNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 4C is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesized 2' -F-DNA using RNA as template without Mn ²⁺ added; lanes 6-8, from left to right, are the products of the synthesis of 2' -F-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using RNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 4D is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesized 2' -OMe-DNA using RNA as template without Mn ²⁺ added; lanes 6-8, from left to right, are the products of the synthesis of 2' -OMe-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using RNA as template with the addition of 1mM Mn ²⁺.

In each figure, P is the 15nt primer band position and F is the 30nt full-length DNA product band position. The bands above the full length band position are bands resulting from incomplete denaturation of the product. When the wild-type Pfu DNA polymerase and the mutant strain P1 cannot synthesize a product, the primer may be degraded because it does not remove the exonuclease activity.

As can be seen from FIG. 4A, under the condition that Mn ²⁺ is not added, the mutant strains P1 and P1 exo ^- can synthesize a certain amount of truncated products of DNA by taking RNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing the DNA by taking RNA as the template of the mutant strains P1 and P1 exo ^- is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strains P1 and P1 exo ^- can synthesize a large amount of full-length products of DNA by taking RNA as a template, so that compared with wild Pfu DNA polymerase, the mutant strains P1 and P1 exo ^- have obviously improved activity of synthesizing DNA by taking RNA as a template.

As shown in FIG. 4B, under the condition of not adding Mn ²⁺, the mutant strain P1 exo ^- can synthesize a small amount of truncated product of RNA by taking RNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing RNA by taking RNA as a template of the mutant strain P1 exo ^- is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strain P1 exo ^- can synthesize a certain amount of truncated products of RNA by taking RNA as a template, so that compared with wild Pfu DNA polymerase, the activity of synthesizing RNA by taking RNA as a template of the mutant strain P1 exo ^- is remarkably improved.

As can be seen from FIG. 4C, under the condition that Mn ²⁺ is not added, the mutant strains P1 and P1 exo ^- can synthesize a certain amount of truncated products of 2'-F-DNA by taking RNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing the 2' -F-DNA by taking RNA as the template is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strains P1 and P1 exo ^- can synthesize a small amount of full-length products of 2'-F-DNA by taking RNA as a template, so that compared with wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing the 2' -F-DNA by taking the RNA as the template is obviously improved.

As shown in FIG. 4D, under the condition that Mn ²⁺ is not added, the mutant strain P1 exo ^- can synthesize a small amount of truncated products of 2'-OMe-DNA by taking RNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing 2' -OMe-DNA by taking RNA as a template of the mutant strain P1 exo ^- is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strain P1 can use RNA as a template to synthesize a small amount of truncated products of 2' -OMe-DNA, and the mutant strain P1 exo ^- can use RNA as a template to synthesize a certain amount of truncated products of 2' -OMe-DNA, so that compared with wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing 2' -OMe-DNA by using RNA as a template is obviously improved.

Example 5

Activity test study of polymerase mutant strains P1, P1 exo ^- for recognition of 2'-F-DNA template specifically, the activities of polymerase mutant strains P1, P1 exo ^- in the present invention were tested by synthesizing DNA, RNA, 2' -F-DNA and 2'-OMe-DNA products using 2' -F-DNA as a template without adding Mn ²⁺ and with the addition of 1mM Mn ²⁺.

The template T30-1 or F-T30 and the complementary primer FAM-P15 are mixed in a ratio of 2:1 in advance, denatured at 95 ℃ for 10min, slowly cooled to room temperature, incubated on ice for 5min, and then the rest reagents are complemented. The DNA, 2' -F-DNA template and the complementary primer sequences are shown in Table 18; the reaction system for the control of the full length product of the synthetic DNA is shown in Table 15; the reaction systems for Pfu polymerase wild type, polymerase mutant P1, P1 exo ^- to synthesize DNA, RNA, 2'-F-DNA and 2' -OMe-DNA are shown in Table 19; the reaction procedure is shown in Table 20. When rNTPs is used as a substrate, 1U/. Mu.L of RNase inhibitor is added into the reaction system. When dNTPs, rNTPs and 2' -F-dNTPs are used as substrates, 1 mu M enzyme (Pfu polymerase wild type, polymerase mutant strain P1 or polymerase mutant strain P1 exo ^-) is added into the reaction system; when 2' -OMe-dNTPs were used as substrates, 2. Mu.M enzyme (Pfu polymerase wild-type, polymerase mutant strain P1 or polymerase mutant strain P1 exo ^-) was added to the reaction system. After the reaction was completed, twice the volume of 2 XTBE-Urea loading buffer was added to the product, denatured at 95℃for 10min, and finally the product was analyzed by 20% denatured polyacrylamide gel electrophoresis.

TABLE 18DNA, 2' -F-DNA templates and complementary primer sequences

/>

F:2' -F modified nucleotides

TABLE 19Pfu DNA polymerase reaction System for synthesizing different nucleic acid molecules Using 2' -F-DNA as template

TABLE 20 reaction procedure for the synthesis of different nucleic acid molecules with DNA, 2' -F-DNA templates

The result of electrophoresis of each reaction product is shown in FIG. 5.

Lane 1 in fig. 5A is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of DNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -F-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -F-DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 5B is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -F-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -F-DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 5C is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesizing 2'-F-DNA using 2' -F-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of 2'-F-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -F-DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 5D is primer; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesized 2'-OMe-DNA using 2' -F-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of 2'-OMe-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -F-DNA as template with the addition of 1mM Mn ²⁺.

As can be seen from fig. 5A, the wild-type Pfu DNA polymerase can synthesize a certain amount of full-length products of DNA using 2' -F-DNA as a template without adding Mn ²⁺, but some truncated products exist, and the mutant strains P1 and P1 exo ^- can synthesize a large amount of full-length products of DNA using 2' -F-DNA as a template, and almost no truncated products exist, so that the activity of synthesizing DNA using 2' -F-DNA as a template of the mutant strains P1 and P1 exo ^- is significantly improved as compared to the wild-type Pfu DNA polymerase; under the condition of adding 1mM Mn ²⁺, the mutant strains P1 and P1 exo ^- can synthesize a large amount of full-length products of DNA by taking 2'-F-DNA as a template, and almost no truncated products exist, so that compared with wild Pfu DNA polymerase, the mutant strains P1 and P1 exo ^- have obviously improved activity of synthesizing DNA by taking 2' -F-DNA as a template.

As can be seen from FIG. 5B, under the condition that Mn ²⁺ is not added, the mutant strains P1 and P1 exo ^- can synthesize a small amount of full-length products of RNA by taking 2'-F-DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing RNA by taking 2' -F-DNA as a template of the mutant strains P1 and P1 exo ^- is remarkably improved; under the condition of adding 1mM Mn ²⁺, wild Pfu DNA polymerase can synthesize a small amount of full-length products of RNA by taking 2' -F-DNA as a template, and mutant strains P1 and P1 exo ^- can synthesize a part of full-length products of RNA by taking 2' -F-DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing RNA by taking 2' -F-DNA as the template is obviously improved.

As can be seen from FIG. 5C, the wild-type Pfu DNA polymerase can synthesize a small amount of the full-length product of 2' -F-DNA by using 2' -F-DNA as a template without adding Mn ²⁺, but some truncated products of 2' -F-DNA exist, and the mutant strains P1 and P1 exo ^- can synthesize a certain amount of the full-length product of 2' -F-DNA by using 2' -F-DNA as a template, so that the activity of synthesizing 2' -F-DNA by using 2' -F-DNA as a template of the mutant strains P1 and P1 exo ^- is remarkably improved compared with the wild-type Pfu DNA polymerase; under the condition of adding 1mM Mn ²⁺, the wild Pfu DNA polymerase can synthesize a small amount of full-length products of 2'-F-DNA by taking 2' -F-DNA as a template, and the mutant strains P1 and P1 exo ^- can synthesize a large amount of full-length products of 2'-F-DNA by taking 2' -F-DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing 2'-F-DNA by taking 2' -F-DNA as a template is remarkably improved.

As can be seen from FIG. 5D, under the condition that Mn ²⁺ is not added, the wild-type Pfu DNA polymerase can synthesize a small amount of truncated products of 2'-OMe-DNA by taking 2' -F-DNA as a template, and the mutant strains P1 and P1 exo ^- can synthesize a large amount of truncated products of 2'-OMe-DNA by taking 2' -F-DNA as a template, so that compared with the wild-type Pfu DNA polymerase, the activity of synthesizing 2'-OMe-DNA by taking 2' -F-DNA as a template of the mutant strains P1 and P1 exo ^- is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strains P1 and P1 exo ^- can synthesize a certain amount of full-length products of 2'-OMe-DNA by taking 2' -F-DNA as a template, so that compared with wild Pfu DNA polymerase, the mutant strains P1 and P1 exo ^- have obviously improved activity of synthesizing 2'-OMe-DNA by taking 2' -F-DNA as a template.

Example 6

Activity test study of polymerase mutant strains P1, P1 exo ^- for recognition of 2'-OMe-DNA template specifically, the activities of polymerase mutant strains P1, P1 exo ^- in the present invention were tested by synthesizing DNA, RNA, 2' -F-DNA and 2'-OMe-DNA products using 2' -OMe-DNA as templates without adding Mn ²⁺ and with the addition of 1mM Mn ²⁺.

The template T30-2 or OMe-T30 and the complementary primer FAM-P18 are mixed in a ratio of 2:1 in advance, denatured at 95 ℃ for 10min, slowly cooled to room temperature, incubated on ice for 5min, and then the rest reagents are complemented. The DNA, 2' -OMe-DNA template and complementary primer sequences are shown in Table 21; the reaction system for the control of the full length product of the synthetic DNA is shown in table 22; the reaction systems for Pfu polymerase wild type, polymerase mutant P1, P1 exo ^- to synthesize DNA, RNA, 2'-F-DNA and 2' -OMe-DNA are shown in Table 23; the reaction procedure is shown in Table 24. When rNTPs is used as a substrate, 1U/. Mu.L of RNase inhibitor is added into the reaction system. When dNTPs, rNTPs and 2' -F-dNTPs are used as substrates, adding 1 mu M enzyme into a reaction system; when 2' -OMe-dNTPs were used as substrates, 2. Mu.M enzyme was added to the reaction system. After the reaction was completed, twice the volume of 2 XTBE-Urea loading buffer was added to the product, denatured at 95℃for 10min, and finally the product was analyzed by 20% denatured polyacrylamide gel electrophoresis.

TABLE 21DNA, 2' -OMe-DNA templates and complementary primer sequences

M:2' -OMe modified nucleotides

Table 22Taq DNA polymerase reaction System for synthesizing full-length DNA product control Using DNA as template

Table 23Pfu DNA polymerase reaction System for synthesizing different nucleic acid molecules Using 2' -OMe-DNA as template

TABLE 24 reaction procedure for the synthesis of different nucleic acid molecules with DNA, 2' -OMe-DNA templates

The result of electrophoresis of each reaction product is shown in FIG. 6.

Lane 1 in fig. 6A is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of DNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -OMe-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -OMe-DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 6B is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of RNA synthesis from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -OMe-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of RNA synthesis by wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -OMe-DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 6C is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesized 2'-F-DNA using 2' -OMe-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of 2'-F-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -OMe-DNA as template with the addition of 1mM Mn ²⁺.

Lane 1 in fig. 6D is a primer control; lane 2 is a DNA product synthesized by Taq DNA polymerase using DNA as a template; lanes 3-5 from left to right are the products of the wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- synthesizing 2'-OMe-DNA using 2' -OMe-DNA as template without addition of Mn ²⁺; lanes 6-8, from left to right, are the products of the synthesis of 2'-OMe-DNA from wild-type Pfu DNA polymerase and its mutants P1, P1 exo ^- using 2' -OMe-DNA as template with the addition of 1mM Mn ²⁺.

In each figure, P is the 18nt primer band position and F is the 30nt full-length DNA product band position. The bands above the full length band position are bands resulting from incomplete denaturation of the product. When the wild-type Pfu DNA polymerase and the mutant strain P1 cannot synthesize a product, the primer may be degraded because it does not remove the exonuclease activity.

As can be seen from fig. 6A, under the condition that Mn ²⁺ is not added, the mutant strain P1 can synthesize a small amount of full-length products of DNA by using 2' -OMe-DNA as a template, and the mutant strain P1 exo ^- can synthesize a large amount of full-length products of DNA by using 2' -OMe-DNA as a template, so that the activity of synthesizing DNA by using 2' -OMe-DNA as a template of mutant strains P1 and P1 exo ^- is significantly improved compared with that of wild-type Pfu DNA polymerase; under the condition of adding 1mM Mn ²⁺, the mutant strain P1 can synthesize a certain amount of full-length products of DNA by taking 2' -OMe-DNA as a template, and the mutant strain P1 exo ^- can synthesize a certain amount of full-length products of DNA by taking 2' -OMe-DNA as a template and almost no truncated products are generated, so that compared with wild Pfu DNA polymerase, the activity of synthesizing DNA by taking 2' -OMe-DNA as a template of the mutant strains P1 and P1 exo ^- is obviously improved.

As can be seen from FIG. 6B, under the condition that Mn ²⁺ is not added, the mutant strain P1 exo ^- can synthesize a small amount of truncated products of RNA by taking 2'-OMe-DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of synthesizing RNA by taking 2' -OMe-DNA as a template of the mutant strain P1 exo ^- is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strain P1 can synthesize a small amount of truncated products of RNA by taking 2' -OMe-DNA as a template, and the mutant strain P1 exo ^- can synthesize a large amount of truncated products of RNA by taking 2' -OMe-DNA as a template, so that compared with wild Pfu DNA polymerase, the activity of the mutant strains P1 and P1 exo ^- for synthesizing RNA by taking 2' -OMe-DNA as a template is obviously improved.

As can be seen from fig. 6C, under the condition that Mn ²⁺ is not added, the mutant strain P1 can synthesize a large amount of full-length products of 2'-F-DNA using 2' -OMe-DNA as a template, and almost no truncated products are generated, and the P1 exo ^- can synthesize a certain amount of full-length products of 2'-F-DNA using 2' -OMe-DNA as a template, so that the activity of synthesizing 2'-F-DNA using 2' -OMe-DNA as a template of mutant strains P1 and P1 exo ^- is significantly improved compared with that of wild-type Pfu DNA polymerase; under the condition of adding 1mM Mn ²⁺, the mutant strains P1 and P1 exo ^- can synthesize the full-length product of 2'-F-DNA by taking the 2' -F-DNA as a template, and almost no truncated product exists, so that compared with the wild Pfu DNA polymerase, the mutant strains P1 and P1 exo ^- have obviously improved activity of synthesizing the 2'-F-DNA by taking the 2' -OMe-DNA as the template.

As shown in FIG. 6D, under the condition that Mn ²⁺ is not added, the mutant strain P1 exo ^- can synthesize a large amount of truncated products of 2'-OMe-DNA by taking the 2' -OMe-DNA as a template, so that compared with the wild Pfu DNA polymerase, the activity of the mutant strain P1 exo ^- for synthesizing the 2'-OMe-DNA by taking the 2' -OMe-DNA as the template is obviously improved; under the condition of adding 1mM Mn ²⁺, the mutant strain P1 exo ^- can synthesize a large amount of truncated products of 2'-OMe-DNA by taking the 2' -OMe-DNA as a template, so that compared with wild Pfu DNA polymerase, the activity of the mutant strain P1 exo ^- for synthesizing the 2'-OMe-DNA by taking the 2' -OMe-DNA as the template is obviously improved.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A Pfu DNA polymerase mutant strain P1 is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. A Pfu DNA polymerase mutant strain P1 exo ^- is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.

3. A gene encoding Pfu DNA polymerase mutant strain P1 of claim 1.

4. A gene according to claim 3, wherein the gene sequence is as shown in SEQ ID No. 3.

5. A gene encoding Pfu DNA polymerase mutant strain P1 exo ^- of claim 2.

6. The gene according to claim 5, wherein the gene sequence is shown in SEQ ID NO. 4.

7. An expression cassette or vector comprising the gene of any one of claims 3-6.

8. A recombinant bacterium or recombinant cell comprising the gene of any one of claims 3-6, the expression cassette of claim 7, or the vector of claim 7.

9. Use of the Pfu DNA polymerase mutant strain P1 of claim 1, the Pfu DNA polymerase mutant strain P1 exo ^- of claim 2, the gene of any one of claims 3-6, the expression cassette of claim 7, the vector of claim 7, the recombinant bacterium of claim 8 or the recombinant cell of claim 8 in the synthesis of a non-natural nucleic acid.

10. A kit for synthesizing nucleic acid, comprising Pfu DNA polymerase mutant strain P1 or Pfu DNA polymerase mutant strain P1 exo ^-.