DNA polymerase and use thereof

Abstract

Provided is a DNA polymerase. The DNA polymerase contains (i) an amino acid sequence as shown in SEQ ID NO: 1; or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as shown in SEQ ID NO: 1 and having polymerase activity; or (iii) an amino acid sequence differing from the amino acid sequence as shown in SEQ ID NO: 1 by no more than 20, 15, 10, 5, 4, 3, 2, or 1 amino acids and having polymerase activity.

Description

DNA polymerase and its application TECHNICAL FIELD

[0001] The present application belongs to the field of gene sequencing, and in particular, the present application relates to DNA polymerase and its application. More particularly, the present application relates to a DNA polymerase, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant strain, a kit, a method for obtaining a DNA polymerase, a nucleic acid amplification method, a library construction method, a sequencing method and use. BACKGROUND

[0002] Rolling-circle replication is a special way of DNA replication, which is often found in the replication process of some viruses and plasmids. It is different from the common semi-conservative replication. The process of rolling-circle replication can be summarized as the following steps: initiation, replication, and circulation. The initiation refers to that at the beginning of rolling-circle replication, a special initiation sequence (origin of replication) forms a cut on the DNA, one side of the cut is 3'-OH end, and the other side is a locally free 5' end. This cut can be initiated by a specific enzyme (such as a cleavage enzyme) or a replication initiation protein, so that the DNA polymerase with rolling-circle replication ability starts the polymerization reaction along the 3'-OH end of the cut position; the replication refers to that when the DNA polymerase continuously adds deoxyribonucleotides to synthesize a new DNA strand by polymerization reaction, at the same time, the 5' end cut off from the downstream parent DNA continues to be free to form a single strand, the DNA polymerase continuously synthesizes along the circular template to generate a new DNA strand; the circulation refers to that the synthesized new DNA strand continues to roll until it returns to the initiation sequence. Because the 5' end is unzipped from the ring, at the same time, the circular double-stranded DNA rotates around its axis continuously, and the DNA growing strand with 3'-OH end as primer continuously extends forward with the other circular DNA strand as template, thus it is called rolling-circle replication. In this replication mode, the extension of DNA can continue, and the DNA chain produced can be many times the length of the parent DNA.

[0003] Rolling-circle replication has the characteristics of high efficiency, rapidity and low error accumulation, and is often found in the replication process of some viruses (such as bacteriophages, single-stranded DNA viruses) and plasmids. Through rolling-circle replication, the organism can rapidly produce a large amount of replication products to meet the needs of its life cycle and transmission. However, the DNA polymerase required by the existing rolling-circle replication has poor thermal stability, which makes it difficult to store for a long time, which to some extent limits its application.

[0004] Therefore, there is an urgent need to develop a DNA polymerase with good thermal stability.

[0005] SUMMARY

[0006] The present application aims to solve at least one of the technical problems existing in the prior art.

[0007] The inventors found that the most commonly used phi29 DNA polymerase in rolling circle amplification is a mesophilic enzyme with an optimal reaction temperature of only 30℃, so it is difficult to apply in complex high temperature environment. However, the DNA sequencing library constructed at a higher temperature of 42℃ or above has more uniform coverage of sequencing reads, lower sequencing depth required, and high temperature reaction conditions can improve the synthesis amount of whole genome amplification products, shorten the amplification time, reduce non-specific amplification products to some extent, and improve the specificity of multiplex chain displacement amplification / rolling circle amplification. In addition, due to the poor thermal stability of the existing wild-type polymerase, it is difficult to store for a long time, which makes the product containing the enzyme have higher requirements for transportation and storage. In order to overcome this problem, the inventors found a DNA polymerase with similar functions through de novo mining. The thermal stability of the DNA polymerase disclosed in the present application is significantly higher than that of the same type of DNA polymerase on the market, and it has good thermal stability while having rolling circle amplification ability, and has a large improvement space. Among them, the optimal reaction temperature of the DNA polymerase disclosed in the present application is higher, about 45℃, which is about 15℃ higher than that of the existing wild-type DNA polymerase (phi29 DNA polymerase); the DNA polymerase disclosed in the present application has good thermal stability, and its thermal denaturation midpoint temperature (Tm) is about 9℃ higher than that of the existing wild-type DNA polymerase (phi29 DNA polymerase); in addition, the sequence consistency of the DNA polymerase disclosed in the present application is only 26.34% compared with the existing wild-type phi29 DNA polymerase, and the sequence consistency is low, so the improvement space is huge; the DNA polymerase disclosed in the present application is a new protein, and its sequence has a consistency of less than 42% compared with the sequence of the existing known protein. m

[0008] ​Based on this, the first aspect of the present application provides a DNA polymerase. According to an embodiment of the present application, the DNA polymerase comprises: (i) an amino acid sequence shown in SEQ ID NO: 1; or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared with the amino acid sequence shown in SEQ ID NO: 1 and having a polymerase activity; or (iii) an amino acid sequence having no more than 20, 15, 10, 5, 4, 3, 2, or 1 amino acid difference compared with the amino acid sequence shown in SEQ ID NO: 1 and having a polymerase activity. The polymerase according to the embodiment of the present application has a higher optimal reaction temperature and a higher thermal stability, and has a larger space for modification compared with the existing wild-type DNA polymerase.

[0009] The second aspect of the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the DNA polymerase according to the first aspect of the present application.

[0010] The third aspect of the present application provides an expression vector. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule according to the second aspect of the present application.

[0011] The fourth aspect of the present application provides a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule according to the second aspect of the present application or the expression vector according to the third aspect of the present application. The recombinant cell can be used to effectively express the aforementioned polymerase in the cell under suitable conditions.

[0012] The fifth aspect of the present application provides a recombinant strain. According to an embodiment of the present application, the recombinant strain expresses the DNA polymerase mutant according to the first aspect of the present application. The recombinant strain can be used to effectively express the aforementioned polymerase in the strain under suitable conditions.

[0013] The sixth aspect of the present application provides a kit. According to an embodiment of the present application, the kit comprises the DNA polymerase according to the first aspect of the present application, the nucleic acid molecule according to the second aspect of the present application, the expression vector according to the third aspect of the present application, the recombinant cell according to the fourth aspect of the present application, or the recombinant strain according to the fifth aspect of the present application. The kit according to the embodiment of the present application can be used to amplify nucleic acids at a higher temperature and construct sequencing libraries.

[0014] In a seventh aspect, the present application provides a method for obtaining a DNA polymerase. According to an embodiment of the present application, the method comprises culturing the recombinant cell of the fourth aspect or the recombinant strain of the fifth aspect under conditions suitable for protein expression to obtain the DNA polymerase. According to the method of the embodiment of the present application, a DNA polymerase with improved optimal reaction temperature, improved thermal stability, and greater space for modification can be prepared.

[0015] In an eighth aspect, the present application provides a method for nucleic acid amplification. According to an embodiment of the present application, the method comprises amplifying a nucleic acid template in the presence of amplification primers, dNTPs, and the DNA polymerase of the first aspect to obtain an amplification product. According to the method of the embodiment of the present application, nucleic acid can be amplified at a higher reaction temperature (45°C and above), and non-specific amplification products can be reduced.

[0016] In a ninth aspect, the present application provides a method for constructing a sequencing library. According to an embodiment of the present application, the method comprises amplifying a nucleic acid to be tested using the method of the eighth aspect to obtain an amplification product, and ligating sequencing adapters to the amplification product to obtain a sequencing library. According to the method of the embodiment of the present application, nucleic acid can be amplified at a higher reaction temperature (45°C and above), non-specific amplification products can be reduced, and a sequencing library can be constructed.

[0017] In a tenth aspect, the present application provides use of the DNA polymerase of the first aspect or the kit of the sixth aspect in the preparation of a nucleic acid amplification product or a sequencing-related product.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 is a sequence alignment diagram in Example 1.

[0020] FIG. 2 is a structural model alignment diagram in Example 2.

[0021] FIG. 3 is a protein purification SDS-PAGE gel diagram (12% gel density) in Example 3. Ni column elution 1 to 4, and Q column elution 1 to 3 correspond to eluate collected at different time periods, respectively.

[0022] FIG. 4 is a schematic diagram for detecting polymerase activity using rolling circle amplification in Example 4.

[0023] FIG. 5 is a determination of the optimal temperature of 18°N DNA polymerase in Example 5.

[0024] Figure 6 is a graph of protein thermal denaturation midpoint temperature in Example 6.

[0025] Figure 7 is a graph of residual activity of phi29 DNA polymerase and 18°N DNA polymerase after heat treatment at 50°C for different time in Example 7.

[0026] Figure 8 is a SDS-PAGE gel electrophoresis of 18°N DNA polymerase Mut 6 mutant (single point mutation V310I) in Example 10. Eluate 1 to 4 from nickel column and eluate 1 to 2 from Q column correspond to eluate collected at different time period.

[0027] Figure 9 is a graph of Q30 ratio as a function of sequencing cycle for single-end sequencing of 100 cycles using DNB prepared from different enzymes in Example 11. DETAILED DESCRIPTION

[0028] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0029] It should be noted that the terms "first", "second" are used only for the purpose of description, and should not be understood as indicating or implying relative importance or implying that the indicated technical features are limited to one or more. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0030] In order to make the present application more easily understood, certain technical and scientific terms are defined below. Unless otherwise apparent in the context of the present application, all other technical and scientific terms used in the present application have the meanings commonly understood by one of ordinary skill in the art to which the present application belongs.

[0031] In the present application, the term "comprising" or "including" is an open-ended expression, i.e. including the content indicated by the present application, but not excluding other aspects.

[0032] In the present application, the terms "identity," "homology," or "similarity" are used in the context of comparing amino acid sequences or nucleic acid sequences to a reference sequence to determine the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as determined by conventional methods, see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that are used in alignment and determination of sequence identity including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are also available that utilize these algorithms, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program from Intelligenetics, Mountain View, California.

[0033] In the present application, the term "at least 80% sequence identity" refers to at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89.5%, 89.9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity to each reference sequence.

[0034] In the present application, the term "Multiple Displacement Amplification (MDA)" is a DNA polymerase-based amplification technique used to amplify and amplify DNA samples in vitro. It is a special whole genome amplification method that can amplify the entire genome or large fragments of DNA without the need for prior specific primer design for the target sequence. MDA uses DNA polymerase with strand displacement activity to amplify under the guidance of off-site primers. In the reaction system, DNA polymerase binds to the DNA fragment on the off-site primer and starts to synthesize new DNA strands. On the synthesized new chain, DNA polymerase continuously amplifies along the template DNA, while the generated new chain is used as a template for continuous strand displacement and amplification. The characteristics of MDA include: (1) highly isothermal reaction: MDA reaction is carried out under isothermal conditions, usually at a temperature of 30-37 degrees Celsius, without the need for complex temperature cycling, simplifying experimental operation; (2) high amplification degree: due to the process of strand displacement, a DNA molecule can produce a large amount of amplification product, thereby achieving high amplification; (3) high specificity: due to the guidance of off-site primers, MDA reaction has high inhibition ability to non-specific primer binding and amplification, which can reduce the generation of non-specific amplification products.

[0035] The present application proposes a DNA polymerase, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant strain, a kit, a method for obtaining a DNA polymerase, a nucleic acid amplification method, a library construction method, a sequencing method and use, which will be described in detail below.

[0036] DNA polymerase

[0037] In a first aspect, the present application provides a DNA polymerase. According to embodiments of the present application, the DNA polymerase comprises: (i) an amino acid sequence as set forth in SEQ ID NO: 1; or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 and having polymerase activity; or (iii) an amino acid sequence having no more than 20, 15, 10, 5, 4, 3, 2, or 1 amino acid difference to the amino acid sequence as set forth in SEQ ID NO: 1 and having polymerase activity. According to embodiments of the present application, the polymerase has a higher optimal reaction temperature, a higher thermal stability, and a larger space for modification, as compared to existing wild-type DNA polymerases.

[0038] According to embodiments of the present application, the amino acid difference comprises an amino acid substitution, a deletion, and / or an insertion, or an N-terminal and / or C-terminal extension.

[0039] According to embodiments of the present application, the amino acid substitution is a point mutation. According to embodiments of the present application, the polymerase has a higher optimal reaction temperature, a higher thermal stability, and a larger space for modification, as compared to existing wild-type DNA polymerases.

[0040] According to embodiments of the present application, the DNA polymerase has a mutation at the V310 site, as compared to the amino acid sequence as set forth in SEQ ID NO: 1. According to embodiments of the present application, the polymerase has a larger space for modification, a further improved thermal stability, and a further improved polymerase activity, as compared to existing wild-type DNA polymerases.

[0041] According to embodiments of the present application, the DNA polymerase has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of the amino acid other than V310, as compared to the amino acid sequence as set forth in SEQ ID NO: 1. According to embodiments of the present application, the polymerase has a larger space for modification, a further improved thermal stability, and a further improved polymerase activity, as compared to existing wild-type DNA polymerases.

[0042] According to an embodiment of the present application, the DNA polymerase has at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, except for the sequence of amino acids other than V310. The polymerase according to the embodiment of the present application has a larger space for modification, further improved thermal stability, and further improved polymerase activity compared to the existing wild-type DNA polymerase.

[0043] According to an embodiment of the present application, the DNA polymerase has at least one mutation of S163, I210, R258, M265, V310, S472, Y97, I4, L64, N80, L157, E183, L334, Y391, K470, N496 compared to the amino acid sequence set forth in SEQ ID NO: 1.

[0044] According to an embodiment of the present application, the DNA polymerase has at least one mutation of S163K, I210V, R258K, M265Y, V310I, V310T, V310L, V310Q, V310M, V310S, V310A, S472K, Y97L, I4R, L64R, N80K, Y97H, L157T, E183K, L334Q, Y391T, K470P, N496T compared to the amino acid sequence set forth in SEQ ID NO: 1. The polymerase according to the embodiment of the present application has a larger space for modification, further improved thermal stability, or further improved polymerase activity compared to the existing wild-type DNA polymerase.

[0045] According to an embodiment of the present application, the DNA polymerase has a V310I mutation compared to the amino acid sequence set forth in SEQ ID NO: 1. The polymerase according to the embodiment of the present application has a larger space for modification, further improved thermal stability, and further improved polymerase activity compared to the existing wild-type DNA polymerase.

[0046] According to an embodiment of the present application, the DNA polymerase has an amino acid sequence as set forth in SEQ ID NO: 2.

[0047] According to an embodiment of the present application, the DNA polymerase has at least one mutation combination of any one of (1) to (25) compared to the amino acid sequence set forth in SEQ ID NO: 1.

[0048] (1) S163K;

[0049] (2) I210V;

[0050] (3) R258K;

[0051] (4) M265Y;

[0052] (5) V310I;

[0053] (6) V310T;

[0054] (7) V310L;

[0055] (8) V310Q;

[0056] (9) V310M;

[0057] (10) V310S;

[0058] (11) V310A;

[0059] (12) S472K;

[0060] (13) V310I+Y97L;

[0061] (14) V310I+I210V;

[0062] (15) V310I+M265Y;

[0063] (16) V310I+I4R;

[0064] (17) V310I+L46R;

[0065] (18) V310I+N80K;

[0066] (19) V310I+Y97H;

[0067] (20) V310I+L157T;

[0068] (21) V310I+E183K;

[0069] (22) V310I+L334Q;

[0070] (23) V310I+Y391T;

[0071] (24) V310I+K470P;

[0072] (25) V310I+N496T.

[0073] The polymerase according to the embodiments of the present application has a larger space for modification, further improved thermal stability and further improved polymerase activity compared with the existing wild-type DNA polymerase.

[0074] According to an embodiment of the present application, the DNA polymerase has any one of mutations of (1) to (6), (8), (10), (12) to (14), (16) to (21), and (25) compared with the amino acid sequence shown in SEQ ID NO: 1. The polymerase according to the embodiment of the present application has a larger space for modification and further improved thermal stability compared with the existing wild-type DNA polymerase.

[0075] According to an embodiment of the present application, the DNA polymerase has any one of mutations of (4) to (6), (10), (15), (17) to (21), and (23) to (25) compared with the amino acid sequence shown in SEQ ID NO: 1. The polymerase according to the embodiment of the present application has a larger space for modification and further improved polymerase activity compared with the existing wild-type DNA polymerase.

[0076] According to an embodiment of the present application, the DNA polymerase has any one of mutations of (4) to (6), (10), (17) to (21), and (25) compared with the amino acid sequence shown in SEQ ID NO: 1. The polymerase according to the embodiment of the present application has a larger space for modification and further improved polymerase activity compared with the existing wild-type DNA polymerase.

[0077] Nucleic acid molecule

[0078] According to a second aspect of the present application, a nucleic acid molecule is provided. According to an embodiment of the present application, the nucleic acid molecule encodes the DNA polymerase according to the first aspect of the present application.

[0079] Expression vector

[0080] According to a third aspect of the present application, an expression vector is provided. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule according to the second aspect of the present application.

[0081] According to an embodiment of the present application, the expression vector is a non-pathogenic viral vector; the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.

[0082] Recombinant cell

[0083] According to a fourth aspect of the present application, a recombinant cell is provided. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule according to the second aspect of the present application or the expression vector according to the third aspect of the present application. The recombinant cell can effectively express the aforementioned polymerase in the cell under suitable conditions.

[0084] According to an embodiment of the present application, the recombinant cell is a prokaryotic cell or a eukaryotic cell.

[0085] According to embodiments of the present application, the recombinant cell is selected from the group consisting of E. coli, yeast, and mammalian cell.

[0086] Recombinant strain

[0087] According to a fifth aspect of the present application, a recombinant strain is provided. According to embodiments of the present application, the recombinant strain expresses the DNA polymerase mutant of the first aspect of the present application. The recombinant strain can be used to express the aforementioned polymerase in the strain under suitable conditions.

[0088] Kit

[0089] According to a sixth aspect of the present application, a kit is provided. According to embodiments of the present application, the kit comprises the DNA polymerase of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the expression vector of the third aspect of the present application, the recombinant cell of the fourth aspect of the present application, or the recombinant strain of the fifth aspect of the present application. The kit of embodiments of the present application can be used to amplify nucleic acids at higher temperatures and construct sequencing libraries.

[0090] According to embodiments of the present application, the kit further comprises at least one of the following components: amplification primers, a nucleic acid template, dNTPs, and a reaction buffer.

[0091] Method for obtaining DNA polymerase

[0092] According to a seventh aspect of the present application, a method for obtaining DNA polymerase is provided. According to embodiments of the present application, the method comprises culturing the recombinant cell of the fourth aspect of the present application or the recombinant strain of the fifth aspect of the present application under conditions suitable for protein expression, so as to obtain the DNA polymerase. The method of embodiments of the present application can be used to prepare DNA polymerases with improved optimal reaction temperature, improved thermal stability, and greater space for modification.

[0093] Nucleic acid amplification method

[0094] According to an eighth aspect of the present application, a nucleic acid amplification method is provided. According to embodiments of the present application, the method comprises amplifying a nucleic acid template in the presence of amplification primers, dNTPs, and the DNA polymerase of the first aspect of the present application, so as to obtain an amplification product. The method of embodiments of the present application can be used to amplify nucleic acids at higher reaction temperatures (45°C and above) and reduce non-specific amplification products.

[0095] According to embodiments of the present application, the amplification is performed by at least one of the following methods: rolling circle amplification, and strand displacement amplification.

[0096] Method for constructing library

[0097] In a ninth aspect, the present application provides a method for constructing a sequencing library. According to an embodiment of the present application, the method comprises: amplifying the nucleic acid to be tested by the method of the eighth aspect of the present application to obtain an amplification product; and ligating a sequencing adaptor to the amplification product to obtain a sequencing library. The method according to the embodiment of the present application can amplify nucleic acid at a higher reaction temperature (45℃ and above), reduce non-specific amplification products, and construct a sequencing library.

[0098] According to an embodiment of the present application, the amplification is performed by at least one of the following methods: rolling circle amplification, strand displacement amplification.

[0099] The sequencing library of the present application can reduce the sequencing depth required in subsequent NGS sequencing, while the coverage of sequencing is more uniform. The "sequencing depth" is generally used to describe the coverage of a genome or a gene region in the sequencing process. It is usually expressed by the average number of times each base is sequenced, or by the ratio of the total number of bases (bp) obtained by sequencing to the genome size (Genome). The coverage refers to the proportion of the sequence obtained by sequencing to the entire target sequence such as the genome sequence. Under high temperature conditions, the 18°N DNA polymerase of the present application can more effectively amplify DNA of complex templates with high GC content and palindromic structure, which means that more target DNA sequences are amplified when constructing a DNA library. Therefore, the coverage is improved during sequencing, thereby reducing the sequencing depth, reducing the resources and costs required for sequencing, and improving the sequencing efficiency. In addition, more uniform coverage also helps to accurately detect and identify genetic variations or sequence polymorphisms in samples.

[0100] Use

[0101] In a tenth aspect, the present application provides the use of the DNA polymerase of the first aspect of the present application or the kit of the sixth aspect of the present application in the preparation of nucleic acid amplification products or sequencing-related products.

[0102] The protein sequences and nucleotide sequences used in the present application are shown in Table 1.

[0103] Table 1: Protein sequences and nucleotide sequences

[0104] The schemes of the present application will be explained below in connection with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be considered as limiting the scope of the present application. If a specific technique or condition is not mentioned in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not mentioned, it is a conventional product that can be obtained on the market.

[0105] Example 1: Mining of 18°N DNA polymerase

[0106] (1) Enzyme mining

[0107] The inventors obtained a novel DNA polymerase by analyzing the metagenomic sequencing data from a deep-sea hydrothermal sample from 18°N, 3583 meters deep, which is named 18°N DNA polymerase (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 4). The enzyme has strand displacement activity and can be used for rolling circle amplification reaction, especially for DNB (DNA nanoball) preparation in the sequencing library construction step.

[0108] (2) Sequence alignment

[0109] The 18°N DNA polymerase obtained in step (1) was subjected to sequence alignment with wild-type phi29 DNA polymerase using the Clustal Omega online sequence alignment website, and the results are shown in Figure 1. The alignment results show that the sequence identity of the two is 26.34%. In Figure 1, the symbols “.”, “:” and “*” in the alignment results represent that the corresponding amino acids have certain similarity (the similarity represented by the three symbols increases in turn), and “*” represents that the amino acids at the site are completely identical.

[0110] Example 2: Structure prediction of 18°N DNA polymerase

[0111] The predicted structure model of 18°N DNA polymerase was subjected to structure (PDB ID: 1×H×) alignment with the existing phi29 DNA polymerase using the TM-align online structure alignment website, and the results are shown in Figure 2. The structure alignment results show that the TM-score is 0.78, that is, although the sequence identity of the two is only 26.34%, the three-dimensional structures of the two are highly homologous (TM-score greater than 0.5 can be considered to have similar protein folding topological structures), and they are likely to have similar functional activities.

[0112] Example 3: Expression and purification of 18°N DNA polymerase

[0113] According to the nucleotide sequence of 18 °N DNA polymerase (SEQ ID NO: 2) shown in Table 1, the sequence synthesis was entrusted to Beijing Lihe Huada Gene Technology Co., Ltd., and the sequence was cloned into the pET-28a(+) expression vector, and the cloning site was NdeI and XhoI.

[0114] The above-mentioned recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Tiangen, item number CB105-02) and plated on solid LB containing 25 μg / mL final concentration of kanamycin, and incubated at 37 °C for overnight, which was used for subsequent expression and purification, and the specific steps are as follows:

[0115] (1) 3-6 single colonies with good growth on the plate were picked and inoculated into 50 mL of LB liquid medium, and incubated at 37 °C for 5-7 h, and the OD 600 was 0.8-4.0, and then the above-mentioned bacterial solution was inoculated into 2 L of LB liquid medium containing 25 μg / mL of kanamycin at an inoculation amount of 1%, and incubated at 37 °C for 2-4 h, and the OD 600 was 0.8-1.0, and the original shaker was pre-cooled to 16 °C, and IPTG was added to the medium to make the final concentration 0.5 mM, and the expression was induced in the shaker at 16 °C and 220 rpm for 12-16 h.

[0116] (2) The bacteria were collected by centrifugation at 8000 g for 30 min, and then resuspended with Ni-A buffer at a ratio of 1:10 (1 g of bacteria was used with 10 mL of Ni-A buffer), and the bacteria were broken in an ice bath environment by ultrasonic method, and the broken liquid was centrifuged at 12000 rpm for 60 min at 4 °C, and the supernatant was filtered with a 0.22 μM filter membrane, and the filtrate obtained after filtration was used as the sample for the purification column (containing 18 °N DNA polymerase).

[0117] (3) The above-mentioned sample (containing 18 °N DNA polymerase) was loaded onto the pre-treated Ni column (HisTrap FF Crude, Cytiva, item number 17525501) at a rate of 3 mL / min, and after the loading was completed, the column was washed with Ni-A buffer for 20 column volumes, and then linear elution was performed (Ni-B liquid accounted for 0-70%, 10.5 CV), and the eluted protein was collected when the ultraviolet absorption peak reached 200 mAu, and the collection was stopped when the ultraviolet absorption peak decreased to 400 mAu.

[0118] (4) The above-mentioned collected eluent was loaded onto the pre-treated SP column (HiTrap SP HP, Cytiva, item number 17115201) (the SP column was pre-equilibrated with 41% SP-B buffer for 10 column volumes), and the flow-through was collected when the ultraviolet absorption peak reached 200 mAu.

[0119] (5) The collected SP column flow-through was diluted with 2.33 times the amount of dilution buffer and filtered with a 0.22 pm filter before being used for Q column (HiTrap Q HP, Cytiva, Cat. No. 17115401) purification. Specifically, the sample was loaded onto a pre-conditioned Q column (Cytiva, Cat. No. 17115401) at a flow rate of 5 mL / min, and after loading was complete, the column was washed with SP-A buffer for 20 column volumes, followed by a linear elution (0-70% SP-B buffer, 10.5 CV), and when the UV absorbance peak was greater than 200 mAu, sample collection was started. The purified sample (containing 18°N DNA polymerase) was dialyzed against 2x dialysis buffer to obtain an 18°N DNA polymerase solution, which was subjected to concentration determination and SDS-PAGE gel electrophoresis (the protein marker used was PageRuler TM Pre-stained protein molecular weight marker (Thermo Scientific, Cat. No. 26616)), and finally stored in storage buffer. The results of the SDS-PAGE gel electrophoresis are shown in Figure 3.

[0120] The specific components of the buffers used in the purification process are as follows:

[0121] Ni-A buffer: 20 mM Tris-HCl (2.42 g / L), 500 mM NaCl (29.22 g / L), 20 mM Imidazole (1.36 g / L), 5% Glycerol (62.5 g / L), pH 7.9;

[0122] Ni-B buffer: 20 mM Tris-HCl (2.42 g / L), 500 mM NaCl (29.22 g / L), 500 mM Imidazole (34.04 g / L), 5% Glycerol (62.5 g / L), pH 7.9;

[0123] Dilution buffer: 20 mM Tris-HCl (2.42 g / L), 5% Glycerol (62.5 g / L), pH 7.9;

[0124] SP-A buffer: 20 mM Tris-HCl (2.42 g / L), 150 mM NaCl (8.77 g / L), 5% Glycerol (62.5 g / L), pH 7.9;

[0125] SP-B buffer: 20 mM Tris-HCl (2.42 g / L), 1 M NaCl (58.44 g / L), 5% Glycerol (62.5 g / L), pH 7.9;

[0126] 2x Dialysis Buffer: 23.75 mM Tris-HCl (2.877 g / L), 237.5 mM KCl (17.705 g / L), 1 mM DTT (0.154 g / L), 0.2375 mM EDTA-2Na-2H2O (0.0884 g / L), 5% Glycerol (62.5 g / L), pH 7.5;

[0127] Storage Buffer: 10 mM Tris-HCl (1.2114 g / L), 100 mM KCl (7.455 g / L), 1 mM DTT (0.15425 g / L), 0.1 mM EDTA-2Na-2H2O (0.037224 g / L), 50% Glycerol (625 g / L), pH 7.5.

[0128] Note: All buffers need to be filtered through a 0.22 pm membrane.

[0129] Example 4: 18°N DNA Polymerase Activity Assay

[0130] The activity of 18°N DNA polymerase was determined by rolling circle amplification. The principle is as follows: single-stranded circular DNA with primers bound as substrate, rolling circle replication, then detecting the ssDNA product by Qubit ssDNA Assay Kit kit (Invitrogen, item number Q10212), the schematic diagram is shown in Figure 4.

[0131] The activity determination process is the same as the DNB preparation process, and the reagents used are BGISEQ-500RS high-throughput sequencing reagent kit (PE100) V3.0 of Huada Zhi Zao (MGI, item number A0215). The main reagents used are DNB preparation buffer, DNB polymerase mixed solution I (main component is polymerization reaction buffer, does not contain polymerase), and DNB termination buffer in the reagent kit, and E. coli standard library reagent V3.0 provided by Huada Zhi Zao (item number 1000007738, batch A0215). The specific process is as follows:

[0132] (1) Take out E. coli standard library V3.0, DNB preparation buffer, DNB polymerase mixed solution I, molecular grade water and DNB termination buffer, and place them in an ice box (about 0.5 h). After melting, use a vortex shaker to shake and mix for 5 s, then centrifuge briefly and place on ice for standby.

[0133] (2) Take a 0.2 mL PCR tube, add 40 fmol E. coli standard library V3.0 and 20 μL DNB preparation buffer on ice, then add molecular-grade water to make up to 40 μL. Vortex the reaction mixture, centrifuge for 5 s, and place it in a PCR instrument for reaction. The reaction conditions are as follows: 95℃ for 1 min, 65℃ for 1 min, 40℃ for 1 min, 4℃∞, and hot cap temperature of 105℃.

[0134] (3) Add 40 μL of DNB polymerase mixture I and 4 μL of 18°N DNA polymerase to the above mixture. Mix the reaction mixture with a vortex mixer, centrifuge for 5 seconds in a mini centrifuge, and then immediately place it in a PCR instrument to start the reaction. The reaction conditions are as follows: 45°C for 1 hour, and the temperature of the hot cap is set to 50°C.

[0135] (4) After the reaction is complete, add 20 μL of DNB stop buffer, and mix slowly by pipetting and blowing 5 times with a pipette and wide-mouth tip. ssDNA Assay Kit and The concentration was detected using a Fluorometer (Invitrogen). The result was 10.8 ng / μL, indicating that the 18°N DNA polymerase exhibits strand displacement activity and can be used for rolling circle amplification.

[0136] Example 5: Optimal temperature test of DNA polymerase at 18°N

[0137] Using the same reaction system as in Example 4, the rolling circle amplification activity of 18°N DNA polymerase (concentration 0.22 mg / mL) at different temperatures was tested, and temperature and corresponding reaction activity curves were obtained. The results are shown in Figure 5. The optimal reaction temperature of 18°N DNA polymerase is about 45°C, which is higher than that of wild-type phi29 DNA polymerase (amino acid sequence as shown in SEQ ID NO:3) (optimal temperature 30°C).

[0138] Example 6: Thermostability Test of DNA Polymerase at 18°N

[0139] Using Protein Thermal Shift TM The dye kit (ThermoFisher, catalog number 91216474) was used to test the thermostability of wild-type phi29 DNA polymerase (amino acid sequence shown in SEQ ID NO:3) and 18°N DNA polymerase, and the midpoint temperature of thermal denaturation (T) was determined. mThe Tm value of phi29 DNA polymerase and 18°N DNA polymerase were confirmed to be 46.5°C and 55.3°C, respectively, according to the above experiment. That is, the thermal denaturation midpoint temperature (Tm value) of 18°N DNA polymerase of the present application is about 9°C higher than that of phi29 DNA polymerase. The specific results are shown in Table 2 and Figure 6.

[0140] Table 2: Thermal denaturation midpoint temperature of polymerase

[0141] Example 7: Residual activity test of 18°N DNA polymerase

[0142] After incubating 1.3 mg / mL phi29 DNA polymerase and 1.3 mg / mL 18°N DNA polymerase at 50°C for different time, residual activity test was performed at 35°C. The residual activity test method was as follows: (1) Take E. coli standard library V3.0, DNB preparation buffer, DNB polymerase mixture I, molecular grade water and DNB termination buffer, and place them on the ice box (about 0.5 h). After melting, use vortex shaker to shake and mix for 5 s, then centrifuge briefly and place on the ice box for standby. (2) Take 0.2 mL PCR tube, add 40 fmol E. coli standard library V3.0 on ice, 20 μL DNB preparation buffer, then add molecular grade water to 40 μL, use vortex shaker to shake and mix, mini centrifuge for 5 s, and place in PCR instrument for reaction. The reaction conditions are as follows: 95°C for 1 min, 65°C for 1 min, 40°C for 1 min, 4°C for ∞, and the hot lid temperature is 105°C. (3) Add 40 μL DNB polymerase mixture I to the above mixture, and 4 μL of heat-treated polymerase (set different reaction groups for 50°C incubation for 0 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 min, respectively). Shake and mix the reaction mixture with vortex shaker, centrifuge for 5 s with mini centrifuge, and then place in PCR instrument for reaction immediately. The reaction conditions are as follows: 35°C for 1 h, and the hot lid temperature is set to 40°C. (4) After the reaction is completed, add 20 μL DNB termination buffer, and gently blow and mix 5 times with pipette and wide-bore pipette tip. Then use ssDNA Assay Kit and Fluorometer instrument (Invitrogen) to detect the concentration. Detect the yield of the amplified product obtained in each group, which represents the corresponding activity. After standardization (i.e. the activity of heat treatment divided by the activity of enzyme without heat treatment, expressed as a percentage, which is the residual activity), plot the relative residual activity against the heat treatment time, and the results are shown in Figure 7. ssDNA Assay Kit and Fluorometer instrument (Invitrogen).

[0143] As shown in FIG. 7, phi29 DNA polymerase has no detectable activity after incubation at 50°C for 5 min, while 18°N DNA polymerase still has 80% residual activity after incubation at 50°C for 10 min. By calculating the enzyme half-life, it is known that the half-life of 18°N DNA polymerase at 50°C is about 38 min, while the half-life of phi29 DNA polymerase at 50°C is less than 5 min, i.e., the half-life of 18°N DNA polymerase at 50°C is more than 7 times that of phi29 DNA polymerase.

[0144] Example 8: Preparation of 18°N DNA polymerase mutants

[0145] To further improve the stability and catalytic activity of 18°N DNA polymerase, a series of mutants of 18°N DNA polymerase were constructed. Specifically, the mutant sites were introduced by PCR amplification using the primer pairs (SEQ ID NO: 5-54) shown in Table 1, with the wild-type 18°N DNA polymerase as the template. The PCR reaction system was as follows: 2.5 μL 10x reaction buffer (Promega pfu DNA polymerase buffer, product number M7745), 0.5 μL 10 mM dNTPs, 0.5 μL 30 ng / μL 18°N DNA polymerase recombinant plasmid template (from the recombinant pET-28a(+) containing the 18°N DNA polymerase gene constructed in Example 3), 1 μL of 10 μM upstream and downstream primers containing the mutant site, 0.5 μL Pfu DNA polymerase (Promega, product number M7745), and nuclease-free water to make up the reaction system to 25 μL. The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min, then 16 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 10 min, and finally further reaction at 72°C for 5 min. After adding 1 μL DpnI (NEB, product number R0176V) to the above PCR reaction product and mixing, the mixture was incubated at 37°C for 2 h, 2 μL of the reaction product was transformed into E. coli BL21(DE3) competent cells by heat shock at 42°C for 90 s, and routine plating (LB solid medium containing 25 μg / ml kanamycin) was performed. After the clones were picked and sequenced to confirm that there was no error, the bacterial liquid was cultured for in vitro induction expression, and the expressed bacterial cells were subjected to one-step purification using a nickel column. The induction expression and purification steps were performed as in Example 3. The purified protein was used for stability and functional activity testing.

[0146] Example 9: Thermal stability testing and activity testing of 18°N DNA polymerase mutants

[0147] Protein Thermal ShiftTM The dye kit (ThermoFisher, catalog number 91216474) was used to perform protein thermal denaturation on the 18°N DNA polymerase mutant obtained in Example 8 at the midpoint temperature (T). m The value was determined, and its rolling circle amplification activity was detected at the same time. The specific steps are as follows: (1) Take out E. coli standard library V3.0, DNB preparation buffer, DNB polymerase mixture I, molecular-grade water and DNB termination buffer, place them on an ice box (about 0.5h), wait for them to melt, use a vortex shaker to mix for 5s, then briefly centrifuge and place them on an ice box for later use. (2) Take 0.2mL PCR tube, add 40fmol E. coli standard library V3.0 and 20μL DNB preparation buffer on ice, then add molecular-grade water to 40μL, vortex shaker to mix the reaction mixture, centrifuge for 5s in a mini centrifuge, place it in a PCR instrument for reaction, and the reaction conditions are as follows: 95℃ for 1min, 65℃ for 1min, 40℃ for 1min, 4℃∞, and hot cap temperature 105℃. (3) Add 40 μL DNB polymerase mixture I and 4 μL heat-treated polymerase to the above mixture. Vortex the mixture to mix well, centrifuge for 5 seconds, and then immediately place it in a PCR instrument to start the reaction. The reaction conditions are as follows: 35℃ for 1 hour, and the hot cap temperature is set to 40℃. (4) After the reaction is complete, add 20 μL DNB stop buffer, and slowly pipette and mix 5 times with a pipette tip. ssDNA Assay Kit and Concentration was measured using a Fluorometer (Invitrogen), and the yield of the amplified product obtained represents the corresponding activity. Relative rolling circle amplification activity was defined as 100% of the rolling circle amplification activity of wild-type 18°N DNA polymerase, and mutant activity was defined as the activity relative to the wild-type 18°N DNA polymerase. The test results are summarized in Table 3.

[0148] Table 3: Midpoint temperature of thermal denaturation and relative activity percentage of DNA polymerase mutants at 18°N

[0149] The results above show that multiple mutants, such as S163K, I210V, R258K, M265Y, V310I, V310T, V310Q, V310S, S472K, V310I+Y97L, V310I+I210V, V310I+I4R, V310I+L46R, V310I+N80K, V310I+Y97H, V310I+L157T, V310I+E183K, and V310I+N496T, exhibit varying degrees of improved thermostability compared to the wild-type 18°N DNA polymerase.

[0150] The rolling circle amplification activity of the plurality of mutants, such as M265Y, V310I, V310T, V310S, V310I+M265Y, V310I+L46R, V310I+N80K, V310I+Y97H, V310I+L157T, V310I+E183K, V310I+Y391T, V310I+K470P and V310I+N496T, is significantly improved compared to the wild-type 18°N DNA polymerase. Among them, some mutants have improved stability and activity, such as M265Y, V310I, V310T, V310S, V310I+L46R, V310I+N80K, V310I+Y97H, V310I+L157T, V310I+E183K and V310I+N496T.

[0151] Example 10: Purification of 18°N DNA polymerase Mut 6 mutant

[0152] In this example, the 18°N DNA polymerase Mut 6 mutant (single-point mutation V310I) was purified according to the method described in Example 3, and the purified product was subjected to SDS-PAGE gel electrophoresis. The results of the SDS-PAGE gel electrophoresis of the purified 18°N DNA polymerase Mut 6 mutant sample are shown in Figure 8.

[0153] Example 11: Sequencing library construction using 18°N DNA polymerase Mut 6 mutant

[0154] In this example, the 18°N DNA polymerase Mut 6 mutant (single-point mutation V310I) with good stability and activity was selected, and after expression and purification, sequencing library construction was performed, and the application effect was detected by testing on a BGISEQ-500RS sequencer. The reagents used in the entire sequencing were BGISEQ-500RS high-throughput sequencing reagent kit (PE100) V3.0 produced by Shenzhen Huada Zhi Zao Technology Co., Ltd. and E. coli standard library V3.0.

[0155] (1) DNB preparation

[0156] Experimental and control groups were set up, wherein the experimental group used the Mut 6 mutant for DNB preparation, and the control group used the DNB polymerase mixture II (i.e. commercial phi29 DNA polymerase) in the above reagent kit for DNB preparation.

[0157] DNB preparation buffer, standard library V3.0, DNB polymerase mix I (buffer for rolling circle amplification reaction in library amplification kit, without polymerase) and DNB polymerase mix II were taken out from -20 °C refrigerator, thawed and mixed on ice box, and placed on ice box for standby. Nuclease Free water and DNB termination buffer were taken out from 4 °C refrigerator, and placed on ice box for standby. 20 μL DNB preparation buffer and 40 fmol E. coli standard library V3.0 were added into the labeled eight-tube in turn, and Nuclease Free water was added to 40 μL, and the eight-tube was vortexed for 5 s and then centrifuged for 3 s on a palm centrifuge. The above eight-tube was placed on a PCR instrument, and the following annealing temperature was set: 95 °C for 1 min, 65 °C for 1 min, 40 °C for 1 min, and 4 °C for keeping. After the reaction was completed, the PCR eight-tube was taken out, placed on an ice box, and after the temperature of the eight-tube cover decreased to room temperature, it was centrifuged for a moment on a palm centrifuge, and placed on ice. Then 40 μL DNB polymerase mix I and 4 μL DNB preparation polymerase (among them, DNA polymerase mix II was used for the control group, and 1.3 mg / mL Mut 6 mutant was used for the experimental group) were added into the above eight-tube in turn. The above reaction mixture was placed on a vortex shaker for continuous mixing for 5 s, and centrifuged for 3 s on a palm centrifuge. It was immediately placed on a PCR instrument to start the reaction. The reaction conditions of the control group were set as 30 °C for 20 min and 4 °C for keeping, and the reaction conditions of the experimental group were set as 37 °C for 2 h and 4 °C for keeping. When the above reaction was completed, the eight-tube was taken out and placed on an ice box, and 20 μL DNB termination buffer was immediately added. 100 μL wide-bore gun head was used for mixing for 5-8 times, and then DNB product yield determination was performed by using Qubit ssDNA Assay Kit and Qubit Fluorometer photometer.

[0158] Table 4: DNB preparation

[0159] (2) DNB loading

[0160] The sample loading reagent plate was taken out and thawed at room temperature (about 1 h), and vortexed to mix, and then centrifuged for a moment and placed in a 4 °C refrigerator for standby. DNB loading buffer II was taken out and vortexed to mix, and then centrifuged for a moment and placed on an ice box for standby. 32 μL DNB loading buffer II was added to the above prepared PCR tube containing 98 μL DNB, and gently mixed and then placed in the DNB placement area of the loading system to start loading. After loading was completed, it was placed at room temperature for 30 min and then placed in a 4 °C refrigerator for standby.

[0161] (3) On-machine testing and data analysis

[0162] The DNB prepared by the DNB polymerase mixture II and the DNB prepared by the 18 °N DNA polymerase Mut 6 mutant were sequenced on a BGISEQ-500RS sequencer, and the sequencing strategy was single-end sequencing SE100, 100 cycles, and the sequencing reagent was BGISEQ-500RS high-throughput sequencing reagent kit (PE100) V3.0. After sequencing, the analysis report was downloaded, and the effect of the DNB prepared by the Mut 6 mutant for sequencing was evaluated by the quality proportion distribution.

[0163] (4) Results

[0164] As shown in FIG. 9, the quality value (Q30) of the DNB prepared by the 18 °N DNA polymerase Mut 6 mutant for sequencing was higher than that of the DNB polymerase mixture II developed based on phi29 DNA polymerase. Specifically, the Q30 value of the first sequencing cycle was around 95%, but as the number of sequencing cycles increased, the Q30 of the Mut 6 group gradually increased from the 50th sequencing cycle, and by the 100th sequencing cycle, the Q30 value of the Mut 6 group was 90.3%, while that of the DNB polymerase mixture II group was 86.3%. In addition, the average Q30 value of the Mut 6 group was 93.6% in the 100 cycles of sequencing, which was higher than that of the DNB polymerase mixture II group, which was 92.5%. In summary, the DNB prepared by the 18 °N DNA polymerase Mut 6 mutant can exhibit better sequencing quality.

[0165] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0166] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A DNA polymerase, characterized in that, The DNA polymerase comprises: (i) an amino acid sequence as set forth in SEQ ID NO: 1; or (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 and having polymerase activity; or (iii) an amino acid sequence having no more than 20, 15, 10, 5, 4, 3, 2, or 1 amino acid difference to the amino acid sequence as set forth in SEQ ID NO: 1 and having polymerase activity.

2. The DNA polymerase of claim 1, wherein, The amino acid difference comprises an amino acid substitution, deletion, and / or insertion, or N-terminal and / or C-terminal extension.

3. The DNA polymerase of claim 2, wherein, The amino acid substitution is a point mutation.

4. The DNA polymerase according to any one of claims 2 or 3, characterized in that, The DNA polymerase has a mutation at position V310 compared to the amino acid sequence as set forth in SEQ ID NO:

1.

5. The DNA polymerase of claim 4, wherein, The DNA polymerase has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of the amino acid other than V310 compared to the amino acid sequence as set forth in SEQ ID NO:

1.

6. The DNA polymerase of claim 5, wherein, The DNA polymerase has at least 97%, at least 98%, or at least 99% sequence identity to the sequence of the amino acid other than V310 compared to the amino acid sequence as set forth in SEQ ID NO:

1.

7. The DNA polymerase according to any one of claims 2 or 3, characterized in that, The DNA polymerase has a mutation at at least one of the following positions compared to the amino acid sequence as set forth in SEQ ID NO: 1: S163, I210, R258, M265, V310, S472, Y97, I4, L64, N80, L157, E183, L334, Y391, K470, N496.

8. The DNA polymerase of claim 7, wherein, The DNA polymerase has a mutation at at least one of the following positions compared to the amino acid sequence as set forth in SEQ ID NO: 1: S163K, I210V, R258K, M265Y, V310I, V310T, V310L, V310Q, V310M, V310S, V310A, S472K, Y97L, I4R, L64R, N80K, Y97H, L157T, E183K, L334Q, Y391T, K470P, N496T.

9. The DNA polymerase of claim 4, wherein, The DNA polymerase has a V310I mutation compared to the amino acid sequence as set forth in SEQ ID NO:

1.

10. The DNA polymerase of claim 4, wherein, The DNA polymerase has an amino acid sequence as set forth in SEQ ID NO:

2.

11. The DNA polymerase of claim 8, wherein, The DNA polymerase has a combination of mutations of any one of (1) to (25) compared to the amino acid sequence as set forth in SEQ ID NO: 1: (1) S163K; (2) I210V; (3) R258K; (4) M265Y; (5) V310I; (6) V310T; (7) V310L; (8) V310Q; (9) V310M; (10) V310S; (11) V310A; (12) S472K; (13) V310I+Y97L; (14) V310I+I210V; (15) V310I+M265Y; (16) V310I+I4R; (17) V310I+L46R; (18) V310I+N80K; (19) V310I+Y97H; (20) V310I+L157T; (21) V310I+E183K; (22) V310I+L334Q; (23) V310I+Y391T; (24) V310I+K470P; (25) V310I+N496T.

12. The DNA polymerase of claim 11, wherein, The DNA polymerase has any one of mutations of (1) to (6), (8), (10), (12) to (14), (16) to (21), and (25) compared to the amino acid sequence shown in SEQ ID NO:

1.

13. The DNA polymerase of claim 11, wherein, The DNA polymerase has any one of mutations of (4) to (6), (10), (15), (17) to (21), (23) to (25) compared to the amino acid sequence shown in SEQ ID NO:

1.

14. The DNA polymerase of claim 11, wherein, The DNA polymerase has any one of mutations of (4) to (6), (10), (17) to (21), (23) to (25) compared to the amino acid sequence shown in SEQ ID NO:

1.

15. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the DNA polymerase according to any one of claims 1 to 14.

16. An expression vector comprising the nucleic acid of claim 15. The expression vector comprises the nucleic acid molecule according to claim 15.

17. A recombinant cell, wherein, The recombinant cell carries the nucleic acid molecule according to claim 15 or the expression vector according to claim 16.

18. The recombinant cell of claim 17, wherein, The recombinant cell is a prokaryotic cell or a eukaryotic cell.

19. The recombinant cell of claim 18, wherein, The recombinant cell is selected from the group consisting of E. coli, yeast, and mammalian cells.

20. A recombinant bacterial strain, characterized in that, The recombinant strain expresses the DNA polymerase according to any one of claims 1 to 14.

21. A kit comprising, Comprising: the DNA polymerase according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to claim 16, the recombinant cell according to any one of claims 17 to 19, or the recombinant strain according to claim 20.

22. The kit of claim 21, wherein Further comprising at least one of the following components: amplification primers, a nucleic acid template, dNTPs, and a reaction buffer.

23. A method of obtaining a DNA polymerase, characterized by, Comprising: culturing the recombinant cell according to any one of claims 17 to 19 or the recombinant strain according to claim 20 under conditions suitable for protein expression so as to obtain the DNA polymerase.

24. A method of nucleic acid amplification, comprising: Comprising: amplifying a nucleic acid to be tested using the method according to claim 24 or 25 so as to obtain an amplification product; 25. The method of claim 24, wherein, and 26. A method of constructing a sequencing library, comprising: ​ ​ ​ The amplification products are ligated to sequencing adapters in order to obtain a sequencing library.

27. The method of claim 26, wherein, The amplification is performed by at least one of the following ways: rolling circle amplification, strand displacement amplification.

28. Use of a DNA polymerase according to any one of claims 1 to 14, or a kit according to claim 21 or 22, for the manufacture of a nucleic acid amplification product or a sequencing related product.

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