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 applications Technical Field

[0001] This invention belongs to the field of gene sequencing, specifically, it relates to DNA polymerase and its applications. More specifically, it relates to a DNA polymerase, nucleic acid molecules, expression vectors, recombinant cells, recombinant strains, kits, methods for obtaining DNA polymerase, nucleic acid amplification methods, library construction methods, sequencing methods, and uses. Background Technology

[0002] Rolling-circle replication is a special type of DNA replication commonly found in the replication processes of certain viruses and plasmids. It differs from the more common semi-conservative replication. The process of rolling-circle replication can be summarized in the following steps: initiation, replication, and cycling. In this context, "initiation" refers to the formation of a nick in the DNA at the start of rolling circle replication by a specific origin sequence. One side of the nick is the 3'-OH end, and the other side is a partially free 5' end. This nick can be initiated by a specific enzyme (such as a cleavage enzyme) or a replication initiation protein. Thus, DNA polymerases capable of rolling circle replication begin the polymerization reaction along the 3'-OH end of the nick. "Replication" refers to the process by which DNA polymerase continuously adds deoxyribonucleotides along the nick to synthesize new DNA strands. Simultaneously, the cleaved 5' end of the downstream parental DNA continues to detach and form a single strand. DNA polymerase then continuously synthesizes along the circular template, generating a new DNA strand. "Circulation" refers to the continued rolling of the synthesized DNA strand until it returns to the origin sequence. This is because as the 5' end unwinds downwards from the loop, the circular double-stranded DNA rotates around its axis, and the DNA growth strand using the 3'-OH end as a primer continuously extends forward using another circular DNA strand as a template. Hence, this is called rolling circle replication. In this replication method, DNA can continue to extend indefinitely, producing DNA strands that can be many times the length of the parent DNA unit.

[0003] Rolling circle replication is characterized by its high efficiency, speed, and low error accumulation, and is commonly used in the replication processes of some viruses (such as bacteriophages and single-stranded DNA viruses) and plasmids. Through rolling circle replication, organisms can rapidly produce large quantities of replication products to meet their life cycle and propagation needs. However, the DNA polymerases required for existing rolling circle replication are difficult to store for long periods due to their poor thermostability, which limits their application to some extent.

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

[0005] Summary of the Invention

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

[0007] The inventors discovered that the phi29 DNA polymerase, most commonly used in rolling circle amplification, is a mesophilic enzyme with an optimal reaction temperature of only 30°C, making it difficult to apply in complex high-temperature environments. DNA sequencing libraries constructed at higher temperatures of 42°C and above exhibit more uniform read coverage, require lower sequencing depths, and, moreover, increase the synthesis of whole-genome amplification products while shortening amplification time, reducing non-specific amplification products to some extent, and improving the specificity of multiple strand substitution amplification / rolling circle amplification. Furthermore, existing wild-type polymerases of the same type have poor thermostability, making long-term storage difficult, which places higher demands on the transportation and preservation of products containing this enzyme. To overcome this problem, the inventors discovered a DNA polymerase with similar function through a de novo discovery approach. The DNA polymerase disclosed in this invention has significantly higher thermostability than similar DNA polymerases currently on the market, possessing both rolling circle amplification capability and good thermostability, and has significant potential for modification. The DNA polymerase disclosed in this invention has a relatively high optimal reaction temperature of approximately 45°C, which is about 15°C higher than that of the existing wild-type DNA polymerase (phi29 DNA polymerase). The DNA polymerase disclosed in this invention also exhibits good thermal stability, with a low thermal denaturation midpoint temperature (T0). m The value is about 9°C higher than that of the existing wild-type DNA polymerase (phi29 DNA polymerase); in addition, the DNA polymerase disclosed in this invention has a sequence identity of only 26.34% compared with the existing wild-type phi29 DNA polymerase, which is low and has great potential for modification; the DNA polymerase disclosed in this invention is a novel protein, and its sequence identity with the sequence of existing known proteins is less than 42%.

[0008] Based on this, in a first aspect, the present invention provides a DNA polymerase. According to embodiments of the present invention, the DNA polymerase comprises: (i) the 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO:1 and having polymerase activity. The polymerase according to embodiments of the present invention, compared with existing wild-type DNA polymerases, has an increased optimal reaction temperature, improved thermal stability, and greater potential for modification.

[0009] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the DNA polymerase described in the first aspect of the present invention.

[0010] In a third aspect, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention.

[0011] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the nucleic acid molecule described in the second aspect of the invention or the expression vector described in the third aspect of the invention. Using this recombinant cell, under suitable conditions, the aforementioned polymerase can be efficiently expressed intracellularly.

[0012] In a fifth aspect, the present invention provides a recombinant bacterial strain. According to embodiments of the invention, the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the invention. Using this recombinant strain, under suitable conditions, the aforementioned polymerase can be efficiently expressed within the strain.

[0013] In a sixth aspect, the present invention provides a reagent kit. According to embodiments of the present invention, the reagent kit comprises the DNA polymerase described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the recombinant cells described in the fourth aspect of the present invention, or the recombinant bacterial strain described in the fifth aspect of the present invention. The reagent kit of the present invention can amplify nucleic acids at higher temperatures and construct sequencing libraries.

[0014] In a seventh aspect, the present invention provides a method for obtaining DNA polymerase. According to an embodiment of the present invention, the method comprises: culturing the recombinant cells described in the fourth aspect of the present invention or the recombinant bacterial strain described in the fifth aspect of the present invention under conditions suitable for protein expression to obtain the DNA polymerase. The method according to the embodiments of the present invention can prepare DNA polymerases with improved optimal reaction temperature, improved thermal stability, and greater potential for modification.

[0015] In an eighth aspect, the present invention provides a method for nucleic acid amplification. According to embodiments of the present invention, the method comprises: amplifying a nucleic acid template in the presence of amplification primers, dNTPs, and the DNA polymerase described in the first aspect of the present invention to obtain amplification products. The method according to embodiments of the present invention allows for nucleic acid amplification at higher reaction temperatures (45°C and above), reducing nonspecific amplification products.

[0016] In a ninth aspect, the present invention provides a method for constructing a sequencing library. According to an embodiment of the present invention, the method includes: amplifying the nucleic acid to be tested using the method described in the eighth aspect of the present invention to obtain an amplification product; and ligating the amplification product to sequencing adapters to obtain a sequencing library. The method according to an embodiment of the present invention allows for nucleic acid amplification at higher reaction temperatures (45°C and above), reducing non-specific amplification products and constructing a sequencing library.

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

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] Figure 1 shows the sequence alignment diagram in Example 1.

[0020] Figure 2 is a comparison diagram of the structural models in Example 2.

[0021] Figure 3 shows the SDS-PAGE gel images (12% gel density) of the protein purification in Example 3. Ni column elutions 1 to 4 and Q column elutions 1 to 3 correspond to the eluents collected at different time points.

[0022] Figure 4 is a schematic diagram illustrating the principle of detecting polymerase activity using rolling circle amplification in Example 4.

[0023] Figure 5 shows the optimal temperature for DNA polymerase at 18°N in Example 5.

[0024] Figure 6 shows the midpoint temperature spectrum of protein thermal denaturation in Example 6.

[0025] Figure 7 shows the residual activity curves of phi29 DNA polymerase and 18°N DNA polymerase after heat treatment at 50°C for different times in Example 7.

[0026] Figure 8 shows the SDS-PAGE gel electrophoresis of the 18°N DNA polymerase Mut 6 mutant (single-point mutation V310I) in Example 10. Ni column elutions 1 to 4 and Q column elutions 1 to 2 correspond to the eluents collected at different time points.

[0027] Figure 9 shows the change in the Q30 ratio with sequencing cycles for single-end sequencing of DNB prepared with different enzymes in Example 11 for 100 cycles. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0032] In this invention, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but 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., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0033] In this invention, the term "at least 80% sequence identity" refers to a sequence identity of at least 80% with each reference sequence, which may 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%, or 99.9%.

[0034] In this invention, the term "Multiple Displacement Amplification" (MDA) is a DNA polymerase-based amplification technique used to amplify and enlarge DNA samples in vitro. It is a specialized whole-genome amplification method that can amplify the entire genome or large fragments of DNA without prior primer design for the target sequence. MDA utilizes a DNA polymerase with strand displacement activity, guided by ectopic primers, for amplification. In the reaction system, the DNA polymerase binds to the DNA fragment on the ectopic primer and begins to synthesize a new DNA strand. Along the synthesized new strand, the DNA polymerase continuously amplifies along the template DNA, simultaneously using the generated new strand as a template for repeated strand displacement and amplification. The characteristics of MDA include: (1) Highly isothermal reaction: The MDA reaction is carried out under isothermal conditions, usually at a temperature of 30-37 degrees Celsius, without the need for complex temperature cycling, which simplifies the experimental operation; (2) High amplification: Due to the strand substitution process, a DNA molecule can produce a large number of amplification products, thereby achieving high amplification; (3) High specificity: Due to the guidance of ectopic primers, the MDA reaction has a high inhibitory ability on non-specific primer binding and amplification, which can reduce the generation of non-specific amplification products.

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

[0036] DNA polymerase

[0037] In a first aspect, the present invention provides a DNA polymerase. According to embodiments of the invention, the DNA polymerase comprises: (i) the 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 with the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO:1 and having polymerase activity. The polymerase according to embodiments of the invention, compared to existing wild-type DNA polymerases, exhibits an increased optimal reaction temperature, improved thermal stability, and greater potential for modification.

[0038] According to embodiments of the present invention, the amino acid differences include amino acid substitution, deletion and / or insertion, or N-terminal and / or C-terminal extension.

[0039] According to embodiments of the present invention, the amino acid substitution is a point mutation. The polymerase according to embodiments of the present invention, compared to existing wild-type DNA polymerases, exhibits an increased optimal reaction temperature, improved thermal stability, and greater potential for modification.

[0040] According to an embodiment of the present invention, the DNA polymerase has a mutation at the V310 site compared to the amino acid sequence shown in SEQ ID NO:1. The polymerase according to the embodiments of the present invention, compared to existing wild-type DNA polymerases, has greater potential for modification, further improved thermostability, and further enhanced polymerase activity.

[0041] According to embodiments of the present invention, the DNA polymerase, compared with the amino acid sequence shown in SEQ ID NO:1, 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 in the amino acid sequences other than V310. The polymerase according to embodiments of the present invention, compared with existing wild-type DNA polymerases, has greater potential for modification, further improved thermostability, and further enhanced polymerase activity.

[0042] According to embodiments of the present invention, the DNA polymerase, compared with the amino acid sequence shown in SEQ ID NO:1, has at least 97%, at least 98%, or at least 99% sequence identity in the sequence of amino acids other than V310. The polymerase according to embodiments of the present invention, compared with existing wild-type DNA polymerases, has greater potential for modification, further improved thermostability, and further enhanced polymerase activity.

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

[0044] According to embodiments of the present invention, the DNA polymerase, compared with the amino acid sequence shown in SEQ ID NO:1, has at least one of the following mutations: S163K, I210V, R258K, M265Y, V310I, V310T, V310L, V310Q, V310M, V310S, V310A, S472K, Y97L, I4R, L64R, N80K, Y97H, L157T, E183K, L334Q, Y391T, K470P, N496T. The polymerase according to embodiments of the present invention, compared with existing wild-type DNA polymerases, has greater potential for modification, further improved thermostability, or further enhanced polymerase activity.

[0045] According to an embodiment of the present invention, the DNA polymerase has a V310I mutation compared to the amino acid sequence shown in SEQ ID NO:1. The polymerase according to the embodiments of the present invention, compared to existing wild-type DNA polymerases, has greater potential for modification, further improved thermostability, and further enhanced polymerase activity.

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

[0047] According to an embodiment of the present invention, the DNA polymerase has any one of the following mutation combinations (1) to (25) compared with the amino acid sequence shown 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 embodiments of the present invention has greater potential for modification, further improved thermal stability, and further enhanced polymerase activity compared to existing wild-type DNA polymerases.

[0074] According to an embodiment of the present invention, the DNA polymerase has any one of the following mutations (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. The polymerase according to the embodiment of the present invention has greater potential for modification and further improved thermostability compared to existing wild-type DNA polymerases.

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

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

[0077] Nucleic acid molecules

[0078] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the DNA polymerase described in the first aspect of the present invention.

[0079] expression carrier

[0080] In a third aspect, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention.

[0081] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector; the non-pathogenic viral vector includes an adenovirus vector or a retrovirus vector.

[0082] Recombinant cells

[0083] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the nucleic acid molecule described in the second aspect of the invention or the expression vector described in the third aspect of the invention. Using this recombinant cell, under suitable conditions, the aforementioned polymerase can be efficiently expressed intracellularly.

[0084] According to an embodiment of the present invention, the recombinant cells are prokaryotic cells or eukaryotic cells.

[0085] According to an embodiment of the present invention, the recombinant cells are selected from Escherichia coli, yeast, or mammalian cells.

[0086] Recombinant strains

[0087] In a fifth aspect, the present invention provides a recombinant bacterial strain. According to embodiments of the invention, the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the invention. Using this recombinant strain, under suitable conditions, the aforementioned polymerase can be efficiently expressed within the strain.

[0088] Reagent test kit

[0089] In a sixth aspect, the present invention provides a reagent kit. According to embodiments of the present invention, the reagent kit comprises the DNA polymerase described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the recombinant cells described in the fourth aspect of the present invention, or the recombinant bacterial strain described in the fifth aspect of the present invention. The reagent kit of the present invention can amplify nucleic acids at higher temperatures and construct sequencing libraries.

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

[0091] Methods for obtaining DNA polymerase

[0092] In a seventh aspect, the present invention provides a method for obtaining DNA polymerase. According to an embodiment of the present invention, the method comprises: culturing the recombinant cells described in the fourth aspect of the present invention or the recombinant bacterial strain described in the fifth aspect of the present invention under conditions suitable for protein expression to obtain the DNA polymerase. The method according to the embodiments of the present invention can prepare DNA polymerases with improved optimal reaction temperature, improved thermal stability, and greater potential for modification.

[0093] Nucleic acid amplification methods

[0094] In an eighth aspect, the present invention provides a method for nucleic acid amplification. According to embodiments of the present invention, the method comprises: amplifying a nucleic acid template in the presence of amplification primers, dNTPs, and the DNA polymerase described in the first aspect of the present invention to obtain amplification products. The method according to embodiments of the present invention allows for nucleic acid amplification at higher reaction temperatures (45°C and above), reducing nonspecific amplification products.

[0095] According to an embodiment of the present invention, the amplification process is performed by at least one of the following methods: rolling circle amplification, chain displacement amplification.

[0096] Database construction methods

[0097] In a ninth aspect, the present invention provides a method for constructing a sequencing library. According to an embodiment of the present invention, the method includes: amplifying the nucleic acid to be tested using the method described in the eighth aspect of the present invention to obtain an amplification product; and ligating the amplification product with a sequencing adapter to obtain a sequencing library. The method according to an embodiment of the present invention allows for nucleic acid amplification at higher reaction temperatures (45°C and above), reducing non-specific amplification products and constructing a sequencing library.

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

[0099] The sequencing library of this invention can reduce the required sequencing depth and achieve more uniform sequencing coverage in subsequent NGS sequencing. "Sequencing depth" typically describes the coverage of the genome or gene region during sequencing. It is usually expressed as the average number of times each base is sequenced, or as the ratio of the total number of bases obtained (bp) to the genome size. Coverage refers to the proportion of the sequence obtained to the entire target sequence, such as the genome sequence. Under high-temperature conditions, the 18°N DNA polymerase of this invention can more effectively amplify DNA with complex templates such as high GC content and palindromic structures. This means that more target DNA sequences are amplified during DNA library construction. This improves coverage during sequencing, thereby reducing sequencing depth, reducing the resources and costs required for sequencing, and improving sequencing efficiency. Furthermore, more uniform coverage also helps in the accurate detection and identification of gene variations or sequence polymorphisms in samples.

[0100] use

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

[0102] The protein and nucleotide sequences used in this invention are shown in Table 1.

[0103] Table 1: Protein and nucleotide sequences

[0104] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

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

[0106] (1) Enzyme mining

[0107] The inventors analyzed metagenomic sequencing data from deep-sea hydrothermal vent samples at 18°N latitude and a depth of 3583 meters to obtain a novel DNA polymerase, which they named 18°N DNA polymerase (amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as shown in SEQ ID NO:4). This enzyme possesses strand displacement activity and can be used for rolling circle amplification reactions, particularly for the preparation of DNB (DNA nanospheres) in the sequencing library construction process.

[0108] (2) Sequence alignment

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

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

[0111] The protein structure of 18°N DNA polymerase was predicted using an AlphaFold-based model. The predicted structure was then aligned with the existing phi29 DNA polymerase structure (PDB ID: 1×H×) using the TM-align online alignment website, as shown in Figure 2. The alignment results showed a TM-score of 0.78, indicating that although the sequence similarity was only 26.34%, their three-dimensional structures were highly homologous (a TM-score greater than 0.5 indicates similar protein folding topology), suggesting they likely possess 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 shown in Table 1 (SEQ ID NO:2), the sequence was synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and the sequence was cloned into the pET-28a(+) expression vector at the NdeI and XhoI cloning sites.

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

[0115] (1) Pick 3-6 healthy single colonies from the plate and inoculate them into 50 mL of LB liquid medium. Incubate at 37°C for 5-7 h. OD 600 Once the bacterial culture reaches 0.8-4.0, a 1% inoculum is added to 2L of LB liquid medium containing 25μg / mL kanamycin. The culture is then incubated at 37°C for 2-4 hours until the OD value is reached. 600 To reach a concentration of 0.8-1.0, pre-cool the original shaker to 16°C, add IPTG to the culture medium to a final concentration of 0.5 mM, and induce expression at 220 rpm for 12-16 h in a shaker at 16°C.

[0116] (2) Collect bacterial cells by centrifugation at 8000g for 30min. Then, resuspend the bacterial cells in Ni-A buffer at a ratio of 1:10 (1g of bacterial cells to 10mL of Ni-A buffer). Use sonication to break down the bacterial cells in an ice bath. Centrifuge the lysate at 12000rpm at 4℃ for 60min. Filter the supernatant through a 0.22μM filter membrane. The filtrate obtained after filtration is used as the sample for purification column loading (containing 18°N DNA polymerase).

[0117] (3) Load the above sample (containing 18°N DNA polymerase) into the pretreated Ni column (HisTrap FF Crude, Cytiva, catalog number 17525501) at a rate of 3 mL / min. After loading, continue to wash with Ni-A buffer for 20 column volumes, and then perform linear elution (Ni-B buffer ratio 0-70%, 10.5 CV). Collect the eluted protein when the UV absorption peak reaches 200 mAu and stop collecting when the UV absorption peak drops to 400 mAu.

[0118] (4) Load the collected eluent onto a pretreated SP column (HiTrap SP HP, Cytiva, catalog number 17115201) (the SP column is pre-equilibrated with 10 column volumes of 41% SP-B buffer). Collect the flow-through when the UV absorption peak reaches 200 mAu.

[0119] (5) The collected SP column flow-through solution was diluted 2.33 times with diluent and filtered through a 0.22 μm filter membrane before being used for purification on a Q column (HiTrap Q HP, Cytiva, catalog number 17115401). Specifically, the sample was loaded onto the pretreated Q column (Cytiva, catalog number 17115401) at a flow rate of 5 mL / min. After loading, the column was washed with SP-A buffer for 20 column volumes, followed by linear elution (SP-B buffer concentration 0-70%, 10.5 CV). When the UV absorption peak was greater than 200 mAu, the sample was collected. The purified sample (containing 18°N DNA polymerase) was dialyzed with 2× dialysis buffer to obtain the 18°N DNA polymerase solution, which was then subjected to concentration determination and SDS-PAGE gel electrophoresis (using PageRuler protein marker). TM The pre-stained protein molecular weight standard (Thermo Scientific, catalog number: 26616) was finally stored in storage buffer. The results of SDS-PAGE gel electrophoresis are shown in Figure 3.

[0120] The specific components of the buffer solution used in the purification process are shown below:

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

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

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

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

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

[0126] 2× 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: 10mM Tris-HCl (1.2114g / L), 100mM KCl (7.455g / L), 1mM DTT (0.15425g / L), 0.1mM EDTA-2Na-2H2O (0.037224g / L), 50% Glycerol (625g / L), pH 7.5.

[0128] Note: All buffer solutions must be filtered through a 0.22 μm membrane.

[0129] Example 4: 18°N DNA polymerase activity assay

[0130] The activity of 18°N DNA polymerase was determined using rolling circle amplification. The specific principle is as follows: single-stranded circular DNA bound to primers is used as substrate for rolling circle replication, and the generated ssDNA product is detected by the Qubit ssDNA Assay Kit (Invitrogen, catalog number Q10212). The principle diagram is shown in Figure 4.

[0131] The activity assay procedure is the same as the DNB preparation procedure. The reagents used are the BGISEQ-500RS high-throughput sequencing reagent kit (PE100) V3.0 (MGI, catalog number A0215) from BGI Genomics. The main components used are the DNB preparation buffer, DNB polymerase mixture I (mainly composed of polymerization reaction buffer, without polymerase), and DNB termination buffer from this kit, as well as the E. coli standard library reagent V3.0 (catalog number 1000007738, batch A0215) provided by BGI Genomics. The specific procedure is as follows:

[0132] (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), and after they thaw, use a vortex shaker to mix them for 5s, then briefly centrifuge and place them on an ice box for later use.

[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 wide-mouth pipette 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 above experiments confirmed that the Tm values ​​of phi29 DNA polymerase and 18°N DNA polymerase were 46.5℃ and 55.3℃, respectively. That is, the midpoint temperature (Tm value) of thermal denaturation of the 18°N DNA polymerase of the present invention is about 9℃ higher than that of phi29 DNA polymerase. The specific results are shown in Table 2 and Figure 6.

[0140] Table 2: Midpoint temperature of polymerase thermal denaturation

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

[0142] After incubating 1.3 mg / mL phi29 DNA polymerase and 1.3 mg / mL 18°N DNA polymerase at 50°C for different times, residual activity was tested at 35°C. The residual activity test method was 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 0.5 h), and after thawing, use a vortex shaker to mix for 5 s, then briefly centrifuge and place them on an ice box for later use. (2) Take 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 40 μL, vortex shake the reaction mixture to mix, centrifuge for 5 s in a mini centrifuge, and place it in a 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 hot cap temperature 105°C. (3) Add 40 μL of DNB polymerase mixture I and 4 μL of heat-treated polymerase (different reaction groups were set up by incubating at 50℃ for 0 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min respectively). Vortex the reaction mixture to mix well, centrifuge for 5 s in a mini centrifuge, and then immediately place it in a PCR instrument to start the reaction. The reaction conditions are as follows: 35℃ for 1 h, and the hot cap temperature is set to 40℃. (4) After the reaction is completed, add 20 μL of DNB stop buffer, and slowly mix 5 times with a pipette and wide-mouth tip. ssDNA Assay Kit and Concentration was detected using a Fluorometer (Invitrogen). The yield of amplified products obtained from each group was measured, and this yield represents the corresponding activity. After standardization of the yield (i.e., the activity after heat treatment divided by the enzyme activity before heat treatment, expressed as a percentage, is the residual activity), the relative residual activity was plotted against the heat treatment time, and the results are shown in Figure 7.

[0143] As shown in Figure 7, the phi29 DNA polymerase showed no detectable activity after incubation at 50°C for 5 minutes, while the 18°N DNA polymerase still retained 80% residual activity after incubation at 50°C for 10 minutes. Calculations based on the enzyme half-life indicate that the 18°N DNA polymerase has an activity half-life of approximately 38 minutes at 50°C, while the phi29 DNA polymerase has an activity half-life of less than 5 minutes at 50°C. Therefore, the half-life of the 18°N DNA polymerase of this invention at 50°C is more than 7 times that of the phi29 DNA polymerase.

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

[0145] To further improve the stability and catalytic activity of 18°N DNA polymerase, a series of mutants were constructed for 18°N DNA polymerase. Specifically, using wild-type 18°N DNA polymerase as a template, the mutation site was introduced by PCR amplification using the primer pairs shown in Table 1 (SEQ ID NO: 5-54). The PCR reaction system consisted of: 2.5 μL 10×reaction buffer (Promega pfu DNA polymerase buffer, catalog 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 obtained in Example 3), 1 μL each of 10 μM forward and reverse primers containing the mutation site, 0.5 μL Pfu DNA polymerase (Promega, catalog number M7745), and the reaction system was brought to 25 μL with nuclease-free water. The PCR reaction program was as follows: pre-denaturation at 95℃ for 2 min, followed by 16 cycles of 95℃ for 30 s, 60℃ for 30 s, and 72℃ for 10 min, with a final reaction at 72℃ for 5 min. The PCR product was mixed with 1 μL of DpnI (NEB, catalog number R0176V) and incubated at 37℃ for 2 h. Then, 2 μL of the product was transformed into competent *E. coli* BL21(DE3) cells via a 42℃ heat shock for 90 s process. The cells were plated using standard methods (LB solid medium containing 25 μg / ml kanamycin). After sequencing verification, the cultured bacterial culture was induced for in vitro expression. The expressed cells were purified using a nickel column in a single step. The induction and purification steps were performed as described in Example 3. The purified protein was used for stability and functional activity testing.

[0146] Example 9: Thermostability and Activity Tests of 18°N DNA Polymerase Mutants

[0147] Using 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] Multiple 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, showed significantly enhanced rolling circle amplification activity compared to the wild-type 18°N DNA polymerase. Some of these mutants exhibit both enhanced 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 the 18°N DNA polymerase Mut 6 mutant

[0152] In this embodiment, 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 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 the 18°N DNA polymerase Mut 6 mutant

[0154] In this embodiment, the 18°N DNA polymerase Mut 6 mutant (single-point mutation V310I), exhibiting good stability and activity, was selected. After expression and purification, a sequencing library was constructed, and its application effect was tested on a BGISEQ-500RS sequencer. The reagents used throughout the sequencing process were the BGISEQ-500RS high-throughput sequencing reagent kit (PE100) V3.0 and the E. coli standard library V3.0, both manufactured by BGI Genomics Co., Ltd.

[0155] (1) DNB preparation

[0156] An experimental group and a control group were set up. The experimental group used the Mut 6 mutant for DNB preparation, while the control group used DNB polymerase mixture II (i.e., commercially available phi29 DNA polymerase) from the above kit to prepare DNB.

[0157] Remove DNB preparation buffer, standard library V3.0, DNB polymerase mixture I (buffer for rolling circle amplification in the library amplification kit, without polymerase), and DNB polymerase mixture II from the -20℃ freezer and thaw and mix them on an ice box. Remove Nuclease-Free water and DNB termination buffer from the 4℃ freezer and place them on an ice box. Add 20 μL of DNB preparation buffer and 40 fmol of E. coli standard library V3.0 to the labeled eight-tube set, then bring the volume to 40 μL with Nuclease-Free water. Vortex the tubes for 5 seconds and then briefly centrifuge for 3 seconds using a handheld centrifuge. Place the eight-tube set on a PCR instrument and set the following annealing temperatures: 95℃ for 1 min, 65℃ for 1 min, 40℃ for 1 min, and hold at 4℃. After the reaction, remove the PCR eight-tube set and place it on an ice box. Once the caps have cooled to room temperature, briefly centrifuge the tubes using a handheld centrifuge and place them on ice. Subsequently, 40 μL of DNB polymerase mixture I and 4 μL of DNB preparation polymerase (DNA polymerase mixture II was used for the control group, and 1.3 mg / mL Lmut 6 mutant was used for the experimental group) were added sequentially to the above eight-tube set. The reaction mixture was vortexed for 5 seconds and then briefly centrifuged for 3 seconds. The mixture was immediately placed in a PCR instrument to begin the reaction. The reaction conditions for the control group were 30℃ for 20 min, held at 4℃; the reaction conditions for the experimental group were 37℃ for 2 h, held at 4℃. After the reaction was completed, the eight-tube set was removed and placed on an ice box, and 20 μL of DNB stop buffer was immediately added. The mixture was mixed 5-8 times with a 100 μL wide-mouth pipette tip, and then the DNB product yield was determined using the Qubit ssDNA Assay Kit and a Qubit Fluorometer.

[0158] Table 4: DNB Preparation

[0159] (2) DNB loading

[0160] Remove the sample loading reagent plate and allow it to thaw at room temperature (approximately 1 hour). Vortex to mix thoroughly, briefly centrifuge, and then store at 4°C. Remove DNB loading buffer II, vortex to mix thoroughly, briefly centrifuge, and then store on an ice pack. Add 32 μL of DNB loading buffer II to the PCR tube containing 98 μL of DNB prepared above, mix gently, and place in the DNB placement area of ​​the loading system to begin loading. After loading, incubate at room temperature for 30 minutes, then store at 4°C.

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

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

[0163] (4) Results

[0164] As shown in Figure 9, the sequencing quality (Q30) of DNB prepared using the 18°N DNA polymerase Mut 6 mutant was higher than that of DNB polymerase mixture II, which was developed based on phi29 DNA polymerase. Specifically, the Q30 value was around 95% in the first sequencing cycle, but as the number of sequencing cycles increased, starting from the 50th sequencing cycle, the Q30 of the Mut 6 group gradually exceeded that of the DNB polymerase mixture II group. 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%. Furthermore, over 100 sequencing cycles, the average Q30 value of the Mut 6 group was 93.6%, higher than the 92.5% of the DNB polymerase mixture II group. In conclusion, DNB prepared using the 18°N DNA polymerase Mut 6 mutant exhibits better sequencing quality.

[0165] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

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

Claims

1. A DNA polymerase, characterized in that, The DNA polymerase comprises: (i) The 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 with the amino acid sequence shown in SEQ ID NO:1 and having polymerase activity; or (iii) An amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO:1 by no more than 20, 15, 10, 5, 4, 3, 2 or 1 amino acid and has polymerase activity.

2. The DNA polymerase according to claim 1, characterized in that, The amino acid differences include amino acid substitutions, deletions and / or insertions, or N-terminal and / or C-terminal extensions.

3. The DNA polymerase according to claim 2, characterized in that, The amino acid substitutions are point mutations.

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

1.

5. The DNA polymerase according to claim 4, characterized in that, The DNA polymerase, compared with the amino acid sequence shown in SEQ ID NO:1, 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 in the sequence of amino acids other than V310.

6. The DNA polymerase according to claim 5, characterized in that, The DNA polymerase has at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1, except for V310.

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 sites compared to the amino acid sequence shown 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 according to claim 7, characterized in that, The DNA polymerase has at least one of the following mutations compared to the amino acid sequence shown 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 according to claim 4, characterized in that, The DNA polymerase has a V310I mutation compared to the amino acid sequence shown in SEQ ID NO:

1.

10. The DNA polymerase according to claim 4, characterized in that, The DNA polymerase has the amino acid sequence shown in SEQ ID NO:

2.

11. The DNA polymerase according to claim 8, characterized in that, The DNA polymerase has any one of the following mutation combinations (1) to (25) compared to the amino acid sequence shown 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 according to claim 11, characterized in that, The DNA polymerase has any one of the following mutations compared to the amino acid sequence shown in SEQ ID NO:1: (1)~(6), (8), (10), (12)~(14), (16)~(21), (25).

13. The DNA polymerase according to claim 11, characterized in that, The DNA polymerase has any one of the following mutations compared to the amino acid sequence shown in SEQ ID NO:1: (4) to (6), (10), (15), (17) to (21), (23) to (25).

14. The DNA polymerase according to claim 11, characterized in that, The DNA polymerase has any one of the following mutations (4) to (6), (10), (17) to (21), (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 carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 15.

17. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 15 or the expression vector of claim 16.

18. The recombinant cell according to claim 17, characterized in that, The recombinant cells are prokaryotic or eukaryotic cells.

19. The recombinant cell according to claim 18, characterized in that, The recombinant cells are selected from Escherichia coli, yeast, or 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 reagent kit, characterized in that, include: 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 according to claim 21, characterized in that, It further includes at least one of the following components: Amplification primers, nucleic acid template, dNTPs, and reaction buffer.

23. A method for obtaining DNA polymerase, characterized in that, include: The recombinant cells according to any one of claims 17 to 19 or the recombinant strain according to claim 20 are cultured under conditions suitable for protein expression in order to obtain the DNA polymerase.

24. A method for nucleic acid amplification, characterized in that, include: The nucleic acid template is amplified in the presence of amplification primers, dNTPs, and the DNA polymerase described in any one of claims 1 to 14 to obtain amplification products.

25. The method according to claim 24, characterized in that, The amplification is performed by at least one of the following methods: rolling circle amplification, chain displacement amplification.

26. A method for constructing a sequencing library, characterized in that, include: The nucleic acid to be tested is amplified using the method described in claim 24 or 25 in order to obtain amplification products; as well as The amplification products are ligated with sequencing adapters to obtain sequencing libraries.

27. The method according to claim 26, characterized in that, The amplification is performed by at least one of the following methods: rolling circle amplification, chain displacement amplification.

28. Use of the DNA polymerase according to any one of claims 1 to 14, or the kit according to claim 21 or 22, in the preparation of nucleic acid amplification products or sequencing-related products.

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