DNA polymerases and uses thereof

Abstract

This invention provides a DNA polymerase and its applications. The DNA polymerase comprises: (a) a protein having the sequence shown in SEQ ID NO: 1; (b) a protein having at least one or more amino acids substituted, deleted, and / or added at at least one of the following sites in the sequence shown in SEQ ID NO: 1: D147, E149, L424, Y425, P426, and A503; or (c) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the sequence in (a) or (b) and possessing DNA polymerase activity. This invention addresses the problem of the limited variety of DNA polymerases in the prior art and is applicable to the field of DNA polymerases.

Description

DNA polymerase and its application Technical Field

[0001] The present invention relates to the field of DNA polymerases, and in particular to a DNA polymerase and applications thereof. Background Art

[0002] DNA polymerase is an enzyme that uses DNA as a replication template and four deoxyribonucleotides as substrates, replicating and synthesizing a new DNA chain complementary to the template chain sequence starting from the 5' end. DNA polymerase can add free deoxyribonucleotides to the 3' end of the newly formed chain, thereby extending the newly formed chain in the 5'-3' direction. Some DNA polymerases possess 3'-5' exonuclease activity, which can correct errors in the newly synthesized DNA chain. For mismatched bases generated during PCR amplification, these DNA polymerases can remove the incorrectly added base, reinsert the correct base, and continue replication, thus ensuring the accuracy of amplification.

[0003] DNA polymerases are divided into six families: A, B, C, D, X, and Y. Most of the thermostable DNA polymerases discovered so far belong to either Family A or Family B. Family A thermostable DNA polymerases are derived from eubacteria, such as Taq (Thermus aquaticus), Tth (Thermus thermophilus), Tca (Thermus caldophilus), Tfl (Thermus flavus), and Tfi (Thermus filiformis) from Thermus genus, and Bst (Bacillus stearothemophilis) from Bacillus. Thermostable DNA polymerases belonging to the B family all originate from archaea, such as Tli (Thermococcus litoralis) from the genus Thermococcus, Pfu (Pyrococcus furiosus) and KOD1 (Thermococcus kodacaraensis) from the genus Thermococcus, as well as Pwo (Pyrococcus woesei), Tgo (Thermococcus gorgonarius), Pab (Pyrococcus abyssi), etc.

[0004] Family A DNA polymerases possess both 5'-3' polymerization and 5'-3' exonucleolytic activity. Family B DNA polymerases possess both 5'-3' polymerization and 3'-5' exonucleolytic activity. The primary application of DNA polymerases is in the polymerase chain reaction (PCR). In PCR, DNA polymerases catalyze the replication of complementary daughter DNA strands in vitro using a parent DNA strand as a template and specific primers as extension starting points through denaturation, annealing, and extension. This application places critical demands on DNA polymerases, namely thermal stability, specificity, fidelity, and processivity. Furthermore, specialized applications, such as PCR amplification of rare or specialized samples, place even higher performance demands on DNA polymerases. DNA polymerases must exhibit high thermal stability and high amplification yields, as well as fast extension rates, high amplification specificity, and the ability to amplify low template amounts and samples in challenging environments (e.g., high salt content). Furthermore, high-throughput sequencing based on reversibly blocked modified dNTPs requires DNA polymerases to exhibit polymerization activity towards the modified bases.

[0005] The structure of B-family DNA polymerases is generally divided into five domains: the N-terminal domain, the exonucleolytic domain, the palm domain, the finger domain, and the thumb domain. It is generally believed that DNA polymerase activity is closely associated with the palm, finger, and thumb domains. The palm domain is the catalytic site of the polymerase; the thumb domain interacts with newly synthesized dsDNA and with incoming nucleotides; the finger domain plays a role in template fixation and nucleotide specificity; and the exonucleolytic domain is associated with 3'-5' exonucleolytic activity. These domains of DNA polymerase work closely together to complete the entire DNA amplification process.

[0006] Compared to A-family DNA polymerases (such as Taq DNA polymerase), the 3'-5' exonucleolytic activity (proofreading activity) of B-family DNA polymerases results in lower error rates, making them more suitable for experiments requiring high PCR fidelity, such as gene screening, sequencing, and mutation detection. Currently, the vast majority of commercially available high-fidelity DNA polymerases are based on Pfu DNA polymerase or KOD DNA polymerase, offering limited options and failing to meet the demands of existing technologies.

[0007] Summary of the Invention

[0008] The main purpose of the present invention is to provide a DNA polymerase and its application to solve the problem of limited types of DNA polymerases in the prior art.

[0009] To achieve the above-mentioned object, according to a first aspect of the present invention, a DNA polymerase is provided, comprising any one of the following proteins: (a) a protein having the sequence shown in SEQ ID NO: 1; (b) a protein in which, in the sequence shown in SEQ ID NO: 1, one or more amino acids are substituted, deleted and / or added at least one of the following sites: D147, E149, L424, Y425, P426 and A503; or (c) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the sequence in (a) or (b) and having DNA polymerase activity.

[0010] Furthermore, the DNA polymerase has thermal stability; preferably, the thermal stability of the DNA polymerase is better than that of KOD DNA polymerase or Pfu DNA polymerase; preferably, the DNA polymerase has 3'-5' exonuclease activity.

[0011] Further, in (b), the amino acid type substituted at each site includes at least one of the following: D147A, E149A, L424A / G / H / I / K / L / M / N / Q / S / T / V, Y425A / G / S / T / V, P426I / A / D / G / L / P / S / T / V or A503L / C / D / H / I / M / N / P / Q / R / S / T / V / Y.

[0012] Furthermore, the DNA polymerase includes a protein having a sequence as shown in SEQ ID NO: 3, or a protein having a homology of more than 70% with the sequence as shown in SEQ ID NO: 3, having DNA polymerization activity, and having polymerization activity for modified deoxyribonucleotides; preferably, the modification includes a 3'-O-blocking modification; preferably, the 3'-O-blocking modification includes a 3'-O-azidomethyl modification.

[0013] In order to achieve the above object, according to a second aspect of the present invention, a DNA molecule is provided, which comprises a polynucleotide encoding the above DNA polymerase.

[0014] Furthermore, the DNA molecule has the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0015] In order to achieve the above object, according to the third aspect of the present invention, a recombinant vector is provided, which contains the above DNA molecule.

[0016] In order to achieve the above object, according to the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or the above recombinant vector.

[0017] Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0018] In order to achieve the above object, according to a fifth aspect of the present invention, a PCR kit is provided, which includes the above DNA polymerase.

[0019] Furthermore, the PCR kit further includes any one or more of the following components: 1) a buffer for providing a PCR amplification environment; 2) PCR primers; 3) a reagent for extracting target DNA.

[0020] Furthermore, the buffer comprises 9.5-10.5 mM Tris-HCl, pH 8.2-9.4, 20-80 mM KCl, 1.0-3.0 mM MgCl2, 35-45 mM TMAC, 0.05-0.15% Triton X-100; preferably, the buffer comprises any one or more of the following:

[0021] 9.5~10.5mM Tris-HCl, pH 8.2~8.6, 20~30mM KCl, 1.0~2.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100;

[0022] 9.5~10.5mM Tris-HCl, pH 8.2~8.6, 70~80mM KCl, 1.0~2.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100;

[0023] 9.5~10.5mM Tris-HCl, pH 8.2~8.6, 70~80mM KCl, 2.01~3.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100;

[0024] 9.5~10.5mM Tris-HCl, pH 8.61~9.0, 20~30mM KCl, 1.0~2.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100;

[0025] 9.5~10.5mM Tris-HCl, pH 9.01~9.4, 20~30mM KCl, 1.0~2.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100;

[0026] More preferably, the buffer comprises any one or more of the following:

[0027] 10mM Tris-HCl, pH 8.4, 25mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100;

[0028] 10mM Tris-HCl, pH 8.4, 75mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100;

[0029] 10mM Tris-HCl, pH 8.4, 75mM KCl, 2.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100;

[0030] 10mM Tris-HCl, pH 8.8, 25mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100; or

[0031] 10mM Tris-HCl, pH 9.2, 25mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100.

[0032] In order to achieve the above-mentioned object, according to the sixth aspect of the present invention, there is provided a use of the above-mentioned DNA polymerase or the above-mentioned PCR kit in PCR amplification, library construction or sequencing.

[0033] In order to achieve the above object, according to a seventh aspect of the present invention, a PCR amplification method is provided, which comprises: performing PCR amplification using the above DNA polymerase or the above PCR kit.

[0034] Furthermore, the PCR method comprises: using DNA polymerase to incorporate modified deoxyribonucleotides during the amplification process; preferably, the modification comprises a 3'-O-blocking modification; preferably, the 3'-O-blocking modification comprises a 3'-O-azidomethyl modification.

[0035] In order to achieve the above object, according to an eighth aspect of the present invention, a library construction kit is provided, which includes the above DNA polymerase.

[0036] Furthermore, the library construction kit also includes any one or more of the following components: 1) an adapter sequence for library construction; 2) a buffer for providing a library construction environment; 3) enzymes for library construction, including one or more of DNA shearing enzymes, end repair enzymes or ligases; 4) modified deoxyribonucleotides.

[0037] In order to achieve the above object, according to the ninth aspect of the present invention, a library construction method is provided, which comprises: constructing a library using the above DNA polymerase or the above library construction kit.

[0038] Furthermore, the library construction method includes: using DNA polymerase to perform PCR amplification on the fragments to be sequenced connected to the sequencing adapter to obtain a DNA library; preferably, the library construction method also includes: using DNA polymerase to incorporate modified deoxyribonucleotides during the DNA amplification process.

[0039] In order to achieve the above-mentioned object, according to the tenth aspect of the present invention, a sequencing kit is provided, which comprises the above-mentioned DNA polymerase.

[0040] Furthermore, the sequencing kit may further include any one or more of the following components: 1) primers for complementary pairing with adapter sequences; 2) dideoxynucleotides; 3) dNTPs; 4) nucleic acid probes; 5) enzymes for sequencing, including DNA ligase and / or endonuclease; 6) buffers for eluting nucleic acid probes and / or dNTPs; and 7) modified deoxyribonucleotides.

[0041] In order to achieve the above-mentioned object, according to the eleventh aspect of the present invention, a sequencing method is provided, which comprises: performing sequencing using the above-mentioned DNA polymerase or the above-mentioned sequencing kit.

[0042] Furthermore, the sequencing method includes using DNA polymerase to perform PCR amplification on the sample to be sequenced to obtain a DNA library; sequencing the DNA library using sequencing technology to obtain sequencing results; preferably, the sequencing method also includes: using DNA polymerase to incorporate modified deoxyribonucleotides during the PCR amplification process.

[0043] By applying the technical solution of the present invention, the above-mentioned DNA polymerase sequence is novel, which can provide a new enzyme skeleton for the modification needs of DNA polymerase in different application scenarios. It can be further optimized and modified according to different application scenarios, and has great room for improvement and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0045] FIG1 shows the sequence alignment results of 22°S DNA polymerase, KOD DNA polymerase, and Pfu DNA polymerase according to Example 1 of the present invention.

[0046] FIG2 shows a diagram showing the domain characteristics analysis of 22°S DNA polymerase according to Example 2 of the present invention.

[0047] FIG3 shows the SDS-PAGE electrophoresis results of the purified sample of 22°S DNA polymerase according to Example 3 of the present invention.

[0048] FIG4 shows a schematic diagram of the polymerization activity detection principle according to Example 5 of the present invention.

[0049] FIG5 shows the results of the 22°S DNA polymerase exo-activity test according to Example 6 of the present invention.

[0050] FIG6 shows the agarose gel electrophoresis results of the 22°S DNA polymerase PCR amplification products according to Example 7 of the present invention.

[0051] FIG7 shows the results of SDS-PAGE electrophoresis analysis of a purified sample of 22° S-Mut DNA polymerase according to Example 9 of the present invention.

[0052] FIG8 shows the Cy3-DNA double-stranded template sequence according to Example 10 of the present invention. DETAILED DESCRIPTION

[0053] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0054] As mentioned in the background art, although based on the ever-increasing performance requirements, more and more commercial DNA polymerases have adopted engineered mutants of naturally occurring wild-type DNA polymerases, many patents and documents have described a variety of useful or performance-enhanced DNA polymerases and mutants thereof, with improved catalytic activity, thermal stability and other properties. However, the space for further development and transformation based on currently known DNA polymerases is limited. Therefore, it is of great significance and value to find novel B family DNA polymerases, which can provide more transformation templates to meet the needs of DNA amplification, synthesis, detection, sequencing and other important recombinant DNA technologies. In the present application, the inventors have attempted to develop a novel DNA polymerase, thereby proposing a series of protection schemes of the present application.

[0055] In a first typical embodiment of the present application, a DNA polymerase is provided, comprising any one of the following proteins: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a DNA polymerase mutant, wherein the amino acid sequence of the DNA polymerase mutant is substituted, deleted and / or added with one or more amino acids at at least one of the following sites in SEQ ID NO: 1: D147, E149, L424, Y425, P426, A503, and the DNA polymerase mutant has DNA polymerase activity; or (c) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the protein in (a) or (b) and having DNA polymerase activity.

[0056] The DNA polymerase represented by SEQ ID NO:1 is a novel DNA polymerase derived from sediments in deep-sea hydrothermal vents and is designated 22°S DNA polymerase. The sequence identity of this DNA polymerase to existing commercial DNA polymerases is less than 45%. Compared to existing similar enzymes, the protein sequence of 22°S DNA polymerase has low homology, providing ample room for modification. It also exhibits excellent thermal stability, 5'-3' polymerization activity, 3'-5' exoclease activity, and PCR amplification performance.

[0057] Moreover, the DNA polymerase mutant obtained by simple mutation modification of the above-mentioned 22°S DNA polymerase has polymerization activity for 3'-O-blocked modified deoxyribonucleotides and can be applied to high-throughput sequencing technology based on 3'-O-blocked modified deoxyribonucleotides.

[0058] SEQ ID NO: 1:

[0059] The above amino acid mutations were all experimentally explored in the examples of this application, and all had DNA polymerase activity compared to the parent having the amino acid sequence shown in SEQ ID NO: 1. The above mutation sites include mutations in the exonuclease domain and the finger domain (active site), which can improve the activity of the protein. For mutations away from the active site, the effect on enhancing the activity of the protein is small, and thus a protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or more homology with the above amino acid sequence and having the same ability to improve the specific enzyme activity can be obtained.

[0060] Those skilled in the art can flexibly add commonly used elements in the prior art to the aforementioned DNA polymerases or mutants. Such elements include, but are not limited to, promoters for regulating transcription and translation, molecular tags for protein purification, signal peptides for protein localization, and other known protein sequences in the prior art. Such elements do not affect the activity of the DNA polymerase, including, but not limited to, its ability to polymerize or to polymerize blocking modified deoxyribonucleotides.

[0061] As used herein, homology refers to the "homology" between amino acid sequences or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides in an amino acid sequence or nucleic acid sequence. The homology of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0062] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology and the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the sequence in a).

[0063] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0064] Generally speaking, according to the rules of substitution, replacement, etc., amino acids with similar properties will have similar effects when substituted with each other. For example, in the above protein, conservative amino acid substitutions may occur. "Conservative amino acid substitutions" include but are not limited to:

[0065] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0066] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0067] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0068] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0069] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0070] The "AlphaFold2" used in this application is a protein structure prediction model based on deep learning launched by DeepMind. It uses deep neural networks to analyze and predict protein sequences, thereby predicting the three-dimensional structure of proteins, providing a new tool and method for protein research.

[0071] In a preferred embodiment, in (b), the type of amino acid substituted at each site includes at least one of the following: D147A, E149A, L424A / G / H / I / K / L / M / N / Q / S / T / V, Y425A / G / S / T / V, P426I / A / D / G / L / P / S / T / V or A503L / C / D / H / I / M / N / P / Q / R / S / T / V / Y; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids; " / " means "or", that is, "Y425A / G / S / T / V" means Y425A, Y425G, Y425S, Y425T or Y425V.

[0072] In a preferred embodiment, the DNA polymerase mutant comprises: a protein shown in SEQ ID NO: 3, or a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the protein shown in SEQ ID NO: 3, having DNA polymerase activity and polymerization activity for blocking-modified deoxyribonucleotides; preferably, the 3'-O-blocking modification comprises a 3'-O-azidomethyl modification.

[0073] The protein represented by SEQ ID NO: 3 is a protein having the mutations D147A+E149A+L424A+Y425A+P426I+A503L. In this application, this DNA polymerase mutant is designated as 22°S-Mut DNA polymerase. In this mutant, positions 147 and 149 are located in the exonuclease domain, and positions 424, 425, 426, and 503 are all located in the finger domain.

[0074] SEQ ID NO: 3:

[0075] The DNA polymerase mutants described above exhibit polymerization activity towards modified single bases (including but not limited to 3'-O-blocked modified deoxyribonucleotides). These DNA polymerase mutants can be used to amplify PCR reactions that are difficult for conventional DNA polymerases to perform, thereby enabling applications in high-throughput sequencing and other fields.

[0076] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the above-mentioned DNA polymerase.

[0077] It should be noted that due to the principle of codon degeneracy, the nucleotide sequence of the translated amino acid sequence is not the only constant sequence. Any nucleotide sequence that can encode the amino acid sequence of the above-mentioned DNA polymerase is a nucleic acid sequence within the scope of this patent.

[0078] In a preferred embodiment, the DNA molecule has the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0079] SEQ ID NO: 2:

[0080] SEQ ID NO: 4:

[0081] Furthermore, those skilled in the art can flexibly add nucleic acid sequences for expressing commonly used elements in the prior art near the above-mentioned DNA molecules, including but not limited to promoters for regulating transcription and translation levels, molecular tags for purifying proteins, signal peptides for localizing proteins, and other known sequences in the prior art.

[0082] In a third typical embodiment of the present application, a recombinant vector is provided, wherein the recombinant vector is connected to the above-mentioned DNA molecule.

[0083] The DNA can encode the DNA polymerase and DNA polymerase mutants and can be linked to a recombinant vector to form circular DNA. Both the DNA and the recombinant vector can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the DNA polymerase and DNA polymerase mutants. For different host species of the DNA molecule or recombinant vector, the nucleotide sequence can be flexibly codon-optimized using existing technologies to obtain a nucleotide sequence with higher transcription and translation efficiency.

[0084] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned DNA molecule or recombinant vector.

[0085] In a preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0086] The host cells described above can replicate recombinant vectors within the host cells and transcribe and translate DNA molecules carried by the recombinant vectors, thereby obtaining large amounts of DNA polymerase. DNA polymerase can be obtained by disrupting the host cells, purifying proteins after disruption, or other methods using existing technologies. The host cells are not of plant origin.

[0087] In a fifth typical embodiment of the present application, a PCR kit is provided, which includes the above-mentioned DNA polymerase.

[0088] In a preferred embodiment, the PCR kit further includes any one or more of the following components: 1) a buffer for providing a PCR amplification environment; 2) PCR primers; 3) a reagent for extracting target DNA.

[0089] The above-mentioned PCR primers include but are not limited to universal primers for amplifying specific target DNA, including but not limited to 16S universal amplification primers for prokaryotic bacteria, 18S universal amplification primers for eukaryotic bacteria, ITS universal amplification primers for fungi, or other PCR primers designed for specific organisms or specific target fragments.

[0090] The buffer is a common PCR buffer in the prior art and is used to provide reaction conditions suitable for the DNA polymerase and polymerase mutant. The DNA polymerase and polymerase mutant of the present application are similar to wild-type Pfu DNA polymerase and can perform PCR reactions in the PCR buffer of the prior art. Those skilled in the art can also flexibly optimize the components in the PCR buffer to obtain a buffer more suitable for such DNA polymerase and polymerase mutant.

[0091] The above reagents for extracting target DNA can extract DNA from the target sample and provide an amplification template for the subsequent PCR reaction.

[0092] In a preferred embodiment, the buffer comprises 9.5-10.5 mM Tris-HCl (pH 8.2-9.4), 20-80 mM KCl, 1.0-3.0 mM MgCl2, 35-45 mM TMAC (tetramethylammonium chloride), and 0.05-0.15% Triton X-100; preferably, the buffer comprises any one or more of the following: 9.5-10.5 mM Tris-HCl (pH 8.2-8.6), 20-30 mM KCl, 1.0-2.0 mM MgCl2, 35-45 mM TMAC, and 0.05-0.15% Triton X-100; 9.5-10.5 mM Tris-HCl (pH 8.2-8.6), 70-80 mM KCl, 1.0-2.0 mM MgCl2, 35-45 mM TMAC, 0.05~0.15% Triton Tris-HCl (pH 8.61~9.0), 20~30mM KCl, 1.0~2.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100; 9.5~10.5mM Tris-HCl (pH9.0~9.4), 20~30mM KCl, 1.0~2.0mM MgCl2, 35~45mM TMAC, 0.05~0.15% Triton X-100.

[0093] More preferably, the buffer comprises any one or more of the following:

[0094] 10mM Tris-HCl (pH 8.4), 25mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100;

[0095] 10mM Tris-HCl (pH 8.4), 75mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100;

[0096] 10mM Tris-HCl (pH 8.4), 75mM KCl, 2.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100;

[0097] 10mM Tris-HCl (pH 8.8), 25mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100; or

[0098] 10mM Tris-HCl (pH 9.2), 25mM KCl, 1.5mM MgCl2, 40mM TMAC, 0.1% Triton X-100.

[0099] In the above buffer solution, pH refers to the pH value of the Tris-HCl solution. After obtaining a Tris-HCl solution of a specific pH, KCl, MgCl2, TMAC, Triton X-100 and other components are added to obtain the above buffer solution.

[0100] In a sixth typical embodiment of the present application, a method for using the above-mentioned DNA polymerase or PCR kit in PCR amplification, library construction or sequencing is provided.

[0101] In a seventh typical embodiment of the present application, a PCR amplification method is provided, in which the above-mentioned DNA polymerase or the above-mentioned PCR kit is used to perform PCR amplification.

[0102] In a preferred embodiment, the PCR method comprises: incorporating a modified deoxyribonucleotide using the above-mentioned DNA polymerase during the amplification process; preferably, the modification comprises a 3'-O-blocking modification; preferably, the 3'-O-blocking modification comprises a 3'-O-azidomethyl modification.

[0103] In an eighth typical embodiment of the present application, a library construction kit is provided, wherein the library construction kit includes the above-mentioned DNA polymerase.

[0104] In a preferred embodiment, the library construction kit further includes any one or more of the following components: 1) an adapter sequence for library construction; 2) a buffer for providing a library construction environment; 3) enzymes for library construction, including one or more of DNA shearing enzymes, end repair enzymes, or ligases; 4) modified deoxyribonucleotides.

[0105] Preferably, the above modifications include but are not limited to 3'-O-blocking modifications; preferably, the 3'-O-blocking modifications include but are not limited to 3'-O-azidomethyl modifications.

[0106] In a ninth typical embodiment of the present application, a library construction method is provided, comprising: constructing a library using the above-mentioned DNA polymerase or the above-mentioned library construction kit.

[0107] In a preferred embodiment, the library construction method comprises: performing PCR amplification on the fragments to be sequenced connected to the sequencing adapters using the above-mentioned DNA polymerase to obtain a DNA library; preferably, the library construction method comprises: incorporating modified deoxyribonucleotides using the DNA polymerase during the DNA amplification process; preferably, the library construction method further comprises: fragmenting the genomic DNA to obtain fragmented DNA; performing end-repair on the fragmented DNA to obtain repaired fragmented DNA; and ligating the adapter sequence to the repaired fragmented DNA to obtain the fragments to be sequenced connected to the sequencing adapters; preferably, the above-mentioned modifications include but are not limited to 3'-O-blocking modifications; preferably, the 3'-O-blocking modifications include but are not limited to 3'-O-azidomethyl modifications.

[0108] Using the above-mentioned library construction kit and / or library construction method, the above-mentioned DNA polymerase can be used to PCR amplify and enrich the fragments to be sequenced connected with sequencing adapters (including but not limited to full-length adapters or truncated adapters), providing sufficient samples for subsequent sequencing.

[0109] In a tenth typical embodiment of the present application, a sequencing kit is provided, wherein the sequencing kit includes the above-mentioned DNA polymerase.

[0110] In a preferred embodiment, the sequencing kit further includes any one or more of the following components: 1) primers for complementary pairing with adapter sequences; 2) dideoxynucleotides; 3) dNTPs; 4) nucleic acid probes; 5) enzymes for sequencing, including DNA ligase and / or endonuclease; 6) buffers for eluting nucleic acid probes and / or dNTPs; and 7) modified deoxyribonucleotides.

[0111] Preferably, the above modifications include but are not limited to 3'-O-blocking modifications; preferably, the 3'-O-blocking modifications include but are not limited to 3'-O-azidomethyl modifications.

[0112] In an eleventh typical embodiment of the present application, a sequencing method is provided, comprising: performing sequencing using the above-mentioned DNA polymerase or the above-mentioned sequencing kit.

[0113] In a preferred embodiment, the sequencing method includes: using the above-mentioned DNA polymerase to perform PCR amplification on the sample to be sequenced to obtain a DNA library, and sequencing the DNA library using sequencing technology to obtain sequencing results; preferably, the above-mentioned sequencing method also includes: using the DNA polymerase to incorporate modified deoxyribonucleotides during the PCR amplification process.

[0114] Preferably, the above modifications include but are not limited to 3'-O-blocking modifications; preferably, the 3'-O-blocking modifications include but are not limited to 3'-O-azidomethyl modifications.

[0115] Utilizing the above-mentioned sequencing kit and / or sequencing method, sequencing of the sample to be sequenced can be achieved by methods such as Sanger sequencing, second-generation sequencing or third-generation sequencing in the prior art. In sequencing, using the above-mentioned DNA polymerase of the present application, common PCR reactions in existing sequencing technologies such as emulsion PCR, bridge PCR, and rolling circle amplification can be achieved. The above-mentioned emulsion PCR can refer to the emulsion PCR operation in the Soild sequencing method of ABI, or the emulsion PCR operation in the Roche 454 sequencing method. The above-mentioned bridge PCR can refer to the bridge PCR operation in the Solexa sequencing method of illumina. The above-mentioned rolling circle amplification technology can refer to the method for preparing DNA nanoballs (DNBs) by amplifying the rolling circle amplification technology (RCA) in the MGI sequencing method of BGI.

[0116] The aforementioned DNA polymerases can be used to achieve PCR amplification, library construction, or high-throughput sequencing of target samples or sequences. In particular, using the aforementioned DNA polymerases (DNA polymerase mutants) that have polymerization activity toward modified single bases (including but not limited to 3'-O-blocking modified deoxyribonucleotides), nucleotides containing blocking modifications can be used for DNA polymerization during PCR amplification or library construction, thereby labeling the target sequence. Alternatively, during sequencing, the aforementioned DNA polymerase mutants can be used to amplify DNA containing blocking modified nucleotides, thereby completing sequencing of the DNA containing the blocking modified nucleotides.

[0117] The following will further explain the beneficial effects of the present application in detail with reference to specific examples. In the examples of the present application, the following exploration is conducted:

[0118] (1) The 22°S DNA polymerase sequence gene was synthesized and inserted into the pET-28a(+) vector (with the insertion sites Nde I / Xho I), and heterologously expressed in Escherichia coli BL21(DE3).

[0119] (2) Use nickel column and ion column for purification.

[0120] (3). Through functional activity assay, it was determined that the enzyme has good thermal stability, 5'-3' polymerization activity and 3'-5' exo-cleavage activity.

[0121] (4). Through functional PCR testing, the enzyme can be directly used in PCR reactions.

[0122] (5) Site-directed mutagenesis of 22°S DNA polymerase and induced expression and purification of mutant protein were performed to obtain 22°S-Mut DNA polymerase.

[0123] (6) The polymerization activity of 22°S-Mut DNA polymerase was tested on a microplate reader for 3'-O-blocked modified deoxyribonucleotides.

[0124] Example 1 Sequence Alignment

[0125] The Clustal Omega online sequence alignment website was used to perform a multiple sequence alignment of the 22°S DNA polymerase disclosed in the present invention, the KOD DNA polymerase, and the Pfu DNA polymerase. The alignment results showed that the sequence identities of the 22°S DNA polymerase, the KOD DNA polymerase, and the Pfu DNA polymerase were 40.28% and 42.52%, respectively. The specific alignment results are shown in Figure 1.

[0126] The amino acid sequence of Pfu DNA polymerase is shown in SEQ ID NO: 5, and the amino acid sequence of KOD DNA polymerase is shown in SEQ ID NO: 6.

[0127] SEQ ID NO: 5:

[0128] SEQ ID NO: 6:

[0129] Example 2 Structural Model

[0130] AlphaFold-based protein structure model prediction for this sequence revealed that 22°S DNA polymerase possesses typical domain structures characteristic of B-family DNA polymerases, including an N-terminal domain, exonuclease domain, palm domain, finger domain, and thumb domain. A diagram illustrating the domain structure of 22°S DNA polymerase is shown in Figure 2.

[0131] Example 3 Construction of 22°S DNA polymerase recombinant plasmid and protein expression and purification

[0132] 1.22°S DNA polymerase plasmid construction and transformation

[0133] The expression plasmid pET-28a(+) / 22°S, containing the 22°S DNA polymerase gene sequence, was synthesized and constructed by Changzhou Xinyisheng Life Science Co., Ltd., with cloning sites located at Nde I and Xho I. The pET-28a(+) / 22°S recombinant plasmid was transformed into E. coli BL21(DE3) competent cells (Cat. No. CB105) purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. for subsequent expression and purification. The transformation steps were as follows: 0.5 μL of 50 ng / μL pET-28a(+) / 22°S plasmid was added to 100 μL of BL21(DE3) competent cells, gently flicked to mix, and incubated on ice for 5 minutes. Heat shock was performed at 42°C for 1 minute, followed by another 5 minutes on ice. 400 μL of antibiotic-free LB was added and shaken at 220 rpm at 37°C for 1 hour. 100 μL of the culture was then plated onto a kanamycin-resistant LB plate and incubated at 37°C for 12-16 hours.

[0134] 2.22°S DNA polymerase protein expression and purification

[0135] (1) Pick 3-6 single colonies that grow well on the plate and inoculate them into a 50 / 250mL LB liquid conical flask. Incubate at 37℃ for 5-7h. OD 600 Then, inoculate the above bacterial solution into 2L / 5L LB medium at a 1% inoculation volume and culture at 37℃ for 2-4 hours until the OD 600 Reach 0.6-0.8. Pre-cool the original shaker to 16°C. Add IPTG to the culture medium to a final concentration of 0.5 mM. Induce expression for 12-16 hours in a shaker at 220 rpm at 16°C.

[0136] (2) After induction of expression, all bacterial cultures were centrifuged at 8000g for 10 minutes to collect the cells. Ni column affinity solution A was then added at a ratio of 1:10 to resuspend the cells and the cells were disrupted by ultrasound in an ice bath.

[0137] (3) Heating treatment: Preheat the water bath to 75-80℃, place the broken bacteria into the water bath, shake and mix, use a clean thermometer to check the internal temperature of the bacteria solution. When it reaches 75℃, start timing for 30 minutes. During this period, shake and mix once every 10 minutes to ensure uniform heating.

[0138] (4) The heat-treated ultrasonic crushed liquid was centrifuged at 12000 rpm for 60 min at 4 ° C, and the supernatant was filtered with a 0.22 μM filter membrane as AKTA ( pure TM 25T purification system, Cytiva) purification column loading sample.

[0139] The above sample was loaded onto a pretreated nickel chromatography column (HisTrap FF, 5 mL column) at a flow rate of 2.5 mL / min. After loading, the Ni column affinity solution A was used to rinse for 20 column volumes (20 CV). Subsequently, a linear elution of 10.5 CV with a Ni column affinity solution B ratio of 0-70% was performed. The eluted protein was collected when the UV absorption peak reached 50 mAu, and the collection was stopped when the UV absorption peak dropped to 200 mAu. The collected samples were subjected to SDS-PAGE electrophoresis analysis to determine the protein purity. The results of SDS-PAGE electrophoresis analysis of the 22°S DNA polymerase purified sample are shown in Figure 3.

[0140] (5) The collected protein elution peak samples were dialyzed at 4°C overnight. After dialysis, the protein concentration was determined and stored in storage solution at -20°C for subsequent functional activity analysis. The enzyme storage concentration was 0.18 mg / mL.

[0141] The specific components of the buffer used in the purification process are as follows:

[0142] Ni column affinity solution A: 20 mM Tris-HCl (2.43 g / L), 500 mM NaCl (29.22 g / L), 20 mM Imidazole (1.36 g / L), 5% Glycerol (62.5 g / L), pH 7.5.

[0143] Ni column affinity solution B: 20 ​​mM Tris-HCl (2.43 g / L), 500 mM NaCl (29.22 g / L), 500 mM Imidazole (34.04 g / L), 5% Glycerol (62.5 g / L), pH 7.5@25°C.

[0144] 2× dialysate: 40 mM Tris-HCl (4.86 g / L), 200 mM KCl (14.91 g / L), 2 mM DTT (dithiothreitol, 0.3085 g / L), 0.2 mM EDTA-2Na (0.0744 g / L), 5% Glycerol (62.5 g / L), pH 8.0@25°C.

[0145] Storage solution: 10 mM Tris-HCl (1.21 g / L), 100 mM KCl (7.455 g / L), 1 mM DTT (0.15425 g / L), 0.1 mM EDTA-2Na (0.0372 g / L), 50% Glycerol (625 g / L), pH 7.5@25°C.

[0146] Example 4 Determination of the thermal stability of 22°S DNA polymerase

[0147] Protein Thermal Shift purchased from Thermo Fisher Scientific TM The protein stability of 22°S DNA polymerase was determined using a dye kit (Cat. No. 4461146). KOD DNA polymerase (SEQ ID NO: 6) and Pfu DNA polymerase (SEQ ID NO: 5) were used as controls. The test reaction system consisted of 5 μL Protein Thermal Shift Buffer, 2 μL of the test protein, 2.5 μL 8× Protein Thermal Shift Dye, and 10.5 μL nuclease-free water. The reaction system was mixed and placed in a StepOne TM Real-time fluorescence quantitative PCR system (Applied Biosystems TM ) were subjected to a 25-99°C temperature increase experiment to monitor the changes in ROX fluorescence signal. The results are shown in Table 1.

[0148] Table 1

[0149] Example 5 22°S DNA polymerase polymerization activity assay

[0150] The polymerization activity was determined using Primed M13mp18 single-stranded DNA (NEB, Cat. No. N4040S) as a substrate. The specific principle is shown in Figure 4. In the presence of polymerization activity, the primer on Primed M13ssDNA will extend along the ssDNA in the 5'→3' direction to generate dsDNA. The generated dsDNA can be used to generate dsDNA by Qubit TM Quantitative detection was performed using the dsDNA HS Quantification Kit (Thermo Fisher Scientific, Cat. No. Q32854). A schematic diagram of the polymerization activity assay based on Primed M13ssDNA is shown in Figure 4. The specific reaction system and components are shown in Table 2.

[0151] Table 2

[0152] After the reaction mixture was reacted at 72°C for 5 min, 2 μL of 0.5 M EDTA was added to terminate the reaction. TM dsDNA concentration was determined using the dsDNA HS Quantification Kit. A negative control was performed by replacing 22°S DNA polymerase with an equal volume of enzyme stock solution. The ΔQubit value was calculated by subtracting the Qubit value of the negative control from the Qubit value of the experimental group. The results are shown in Table 3, indicating that 22°S DNA polymerase exhibits polymerization activity at 72°C.

[0153] Table 3

[0154] Example 6 22°S DNA polymerase exo-activity assay

[0155] The exosome activity of 22°S DNA polymerase was qualitatively tested using a terminal mismatch fluorescent probe method. The probe sequences were ATCAGCAGGCCACACGTTAAACTGT-BHQ2 (SEQ ID NO: 7) and FAM-5'-TGTCTTTAAC GTGTGGCCTGCTGAT (SEQ ID NO: 8). The two were mixed in equimolar amounts (final concentration 10 μM) and annealed to serve as the fluorescent probe substrate for the exosome activity test. The reaction system used for the exosome activity test (total volume 25 μL) was as follows: 2.5 μL Reaction buffer, 0.25 μL of 10 μM fluorescent probe substrate, 2 μL of 22°S DNA polymerase (enzyme concentration 0.18 mg / mL), and 20.25 μL of nuclease-free water were used. The positive control group consisted of KOD DNA polymerase. For the negative control group, the enzyme solution was replaced with an equal volume of enzyme stock solution. The reaction system was set up on ice. After preparation, the plate was transferred to a 384-well plate and placed in a microplate reader (BioTek Synergy H1, Agilent) for fluorescence signal detection. The excitation and emission wavelengths were set at 492 nm and 518 nm, respectively. The reaction temperature was 37°C, and fluorescence signals were collected every 30 seconds for a total reaction time of 1 hour. The results of the 22°S DNA polymerase exolytic activity test are shown in Figure 5 (fluorescence values ​​are calculated after subtracting the corresponding blank control values). The exolytic activity of 22°S DNA polymerase is comparable to that of KOD DNA polymerase.

[0156] Example 7 Evaluation of PCR Potential of 22°S DNA Polymerase

[0157] Five representative PCR reaction buffers were used to test the potential of 22°S DNA polymerase for PCR applications. The components of the five reaction buffers used are shown in Table 4.

[0158] Table 4

[0159] The Escherichia coli genome was used as a template to amplify the 16s gene. The upstream and downstream primers used were E16S-27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 9) and E16S-1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 10). The PCR reaction system is shown in Table 5.

[0160] Table 5

[0161] The PCR cycle was as follows: initial denaturation at 95°C for 3 minutes; 30 cycles of 95°C for 20 seconds, 58°C for 15 seconds, and 72°C for 1.5 minutes; and a final extension at 72°C for 7 minutes, followed by storage at 4°C. PCR products were subjected to agarose gel electrophoresis using the Tiangen 100bp DNA ladder (Cat. No. MD109-01) as a molecular marker. The agarose gel electrophoresis results of the PCR amplification products using 22°S DNA polymerase are shown in Figure 6. 22°S DNA polymerase was able to perform PCR amplification in all five buffer conditions, with Buffer 4 providing the best amplification and highest yield.

[0162] Example 8-22°S DNA polymerase mutant vector construction

[0163] Using the wild-type 22°S DNA polymerase expression plasmid pET-28a / 22°S as a template, a mutant containing six mutation sites, 22°S-Mut, was constructed. Mutations were introduced at the corresponding sites through three rounds of rapid PCR amplification using the three primer pairs listed in Table 6.

[0164] The rapid PCR reaction system consisted of 5 μL of Pfu DNA polymerase 10X Buffer with MgSO₄, 1 μL of dNTP Mix (10 mM each), 1.5 μL of forward primer (10 μM), 1.5 μL of reverse primer (10 μM), 50 ng of template DNA, 0.5 μL of Pfu DNA polymerase (3 U / μL, Promega, Cat. No. M7741), and nuclease-free water to a total volume of 50 μL. The rapid PCR reaction program was as follows: initial denaturation at 95°C for 2 min; 16 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 8 min; a final extension at 72°C for 5 min, and storage at 4°C.

[0165] Add 1 μL of DpnI (20 U / μL, NEB, Catalog No. R0176V) to the PCR product and incubate at 37°C in a metal bath for 2 hours to digest the template. The reaction product was then transformed into E. coli DH5α competent cells (Catalog No. CB101) purchased from Tiangen. The transformation steps were as follows: add 10 μL of the reaction product to 100 μL of DH5α competent cells, gently flick to mix, and place on ice for 30 minutes. Heat shock at 42°C for 1 minute, then place on ice again for 10 minutes. Add 400 μL of antibiotic-free LB medium and shake at 37°C at 220 rpm / min for 1 hour. Centrifuge the culture at 3000 g for 3 minutes, discard a portion of the supernatant, and retain approximately 100 μL. Resuspend and mix thoroughly, then spread on a kanamycin-resistant LB plate and incubate at 37°C for 12-16 hours.

[0166] Several single colonies were selected and cultured overnight in a 37°C shaker, followed by plasmid DNA mini-extraction. The plasmids were sent to Beijing Liuhe BGI Genomics Co., Ltd. for Sanger sequencing to verify the correct introduction of the mutation sites. The correctly sequenced plasmid pET-28a / 22°S-Mut, which carries a total of six mutation sites (D147A+E149A+L424A+Y425A+P426I+A503L), was transformed into the expression host Escherichia coli BL21(DE3) competent cells. The transformation process was similar to that in Example 3 to obtain a recombinant E. coli strain expressing the mutant 22°S-Mut.

[0167] Table 6

[0168] Example 9 Expression and Purification of 22°S DNA Polymerase Mutant Protein

[0169] The expression and purification of the 22°S DNA polymerase mutant 22°S-Mut (D147A+E149A+L424A+Y425A+P426I+A503L) were performed as described in Example 3. Protein elution peak samples were collected and analyzed by SDS-PAGE electrophoresis to determine protein purity. The results of SDS-PAGE electrophoresis analysis of the purified 22°S-Mut DNA polymerase sample are shown in Figure 7. 22°S-Mut was stored in storage solution at a stock concentration of 0.25 mg / mL.

[0170] Example 10 Detection of Polymerization Activity of 22°S DNA Polymerase Mutants for Modified Single Base Incorporation

[0171] Using dATP labeled with Cy5 fluorescent dye and modified with a 3'-O-azidomethyl blocking group as a substrate and double-stranded DNA labeled with Cy3 fluorescent dye as a template primer, we simulated the incorporation of blocked modified nucleotides during high-throughput sequencing. The polymerization activity of 22°S-Mut DNA polymerase for single-base incorporation of modified nucleotides was assayed by measuring the FRET-Cy5 fluorescence signal generated when the substrate polymerizes onto the template strand, due to the interaction distance between Cy3 and Cy5.

[0172] The detection method is as follows: 10X reaction buffer consists of 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, pH 8.5 at 25°C. A 50 μL reaction system is prepared as follows: 5 μL 10X reaction buffer, 40 μM dT / G / GTP, 0.1 mg / mL BSA (bovine serum albumin), 1 μM Cy3-double-stranded DNA template (structure shown in Figure 8), 4 μM Cy5-modified 3'-O-AzidoMethyl-dATP (3'-O-azidomethyl-dATP), and 0.5 μg 22°S DNA polymerase or 22°S-Mut DNA polymerase. The reaction system is made up to 50 μL with nuclease-free water.

[0173] The sense strand of the Cy3-DNA double-stranded template is 5'-CGTGTATGCGTAATAGGATCCCGACTCACTA TGGACG-3' (SEQ ID NO: 17); the antisense strand is 5'-CGTGTATCGTCCATAGTGAGTCGGGATC CTATTACGC-3' (SEQ ID NO: 18), and the 5' ends of both the sense and antisense strands are modified with Cy3.

[0174] The reaction system was set up on ice, transferred to a 384-well plate, and placed in a microplate reader (BioTek Synergy H1, Agilent) for fluorescence signal detection. The reaction temperature was 40°C, and fluorescence signals at 530 / 568 nm and 630 / 676 nm (FRET Cy5) were collected every 30 seconds for a total of 2 hours. For the negative control, an equal volume of enzyme stock solution was used to replace 22°S-Mut DNA polymerase. After the reaction, the raw data was exported and the maximum slope (ΔFRET-Cy5 fluorescence per unit time (ΔRFU)) was calculated to characterize the polymerase's polymerization activity for the incorporation of modified single bases.

[0175] The experimental results are shown in Table 7 below. The fluorescence signal curve for the 22°S DNA polymerase experimental group was the same as that for the negative control group, with no FRET Cy5 fluorescence signal detected. However, an increase in fluorescence signal was detected in the 22°S-Mut DNA polymerase experimental group, with a polymerization rate of 1.4ΔRFU / min. The 22°S-Mut DNA polymerase obtained through mutational modification exhibits polymerization activity towards modified single bases. By effectively mutagenizing 22°S DNA polymerase, mutants with great potential for high-throughput sequencing can be generated.

[0176] Table 7

[0177] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: compared with the existing B family DNA polymerase or other DNA polymerases, the above DNA polymerase is a new type of DNA polymerase, and the wild type of the polymerase already has good thermal stability and PCR amplification ability, the original enzymatic performance is good, and PCR amplification can be performed well; the sequence novelty is good, and a new enzyme skeleton can be provided for the modification needs of different application scenarios of DNA polymerase, and further system optimization and modification can be performed according to different application scenarios, with large room for improvement and application potential. Furthermore, by performing simple mutation modification on the above wild type DNA polymerase, a DNA polymerase mutant with polymerization activity for deoxyribonucleotides with blocking modifications such as 3'-O-azidomethyl can be obtained, which has application value in high-throughput sequencing technology based on deoxyribonucleotides with blocking modifications such as 3'-O-azidomethyl.

[0178] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A DNA polymerase, characterized in that, Comprising any one of the following proteins: (a) A protein having the sequence shown in SEQ ID NO: 1; (b) A protein in which at least one of the following sites is substituted, deleted, and / or added with one or several amino acids in the sequence shown in SEQ ID NO: 1: D147, E149, L424, Y425, P426, and A503; or (c) A protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the sequence in (a) or (b) and having DNA polymerase activity.

2. The DNA polymerase according to claim 1, wherein The DNA polymerase has thermal stability; Preferably, the thermal stability of the DNA polymerase is superior to that of KOD DNA polymerase or Pfu DNA polymerase; Preferably, the DNA polymerase has 3'-5' exonuclease activity.

3. The DNA polymerase according to claim 1, wherein, In (b) above, the types of amino acids substituted at each site include at least one of the following: D147A, E149A, L424A / G / H / I / K / L / M / N / Q / S / T / V, Y425A / G / S / T / V, P426I / A / D / G / L / P / S / T / V or A503L / C / D / H / I / M / N / P / Q / R / S / T / V / Y.

4. The DNA polymerase according to any one of claims 1 to 3, characterized in that, The DNA polymerase includes: A protein having the sequence shown in SEQ ID NO: 3, or A protein having more than 70% homology with the sequence shown in SEQ ID NO: 3, having DNA polymerase activity, and having polymerase activity on modified deoxyribonucleotides; Preferably, the modification includes 3'-O-blocking modification; Preferably, the 3'-O-blocking modification includes 3'-O-azidomethyl modification.

5. A DNA molecule, characterized in that, The DNA molecule contains a polynucleotide encoding the DNA polymerase according to any one of claims 1 to 4.

6. The DNA molecule according to claim 5, wherein The DNA molecule has the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO:

4.

7. A recombinant vector, characterized in that, The recombinant vector contains the DNA molecule according to claim 5 or 6.

8. A host cell, characterized in that, The host cell contains the DNA molecule according to claim 5 or 6, or the recombinant vector according to claim 7.

9. The host cell according to claim 8, wherein The host cell includes a prokaryotic cell or a eukaryotic cell; Preferably, the prokaryotic cell includes Escherichia coli.

10. A PCR kit, characterized in that, The PCR kit includes the DNA polymerase according to any one of claims 1 to 4.

11. The PCR kit according to claim 10, wherein The PCR kit further includes any one or more of the following components: 1) A buffer for providing a PCR amplification environment; 2) PCR primers; 3) Reagents for extracting the target DNA.

12. The PCR kit according to claim 11, wherein, The buffer includes 9.5 - 10.5 mM Tris-HCl, pH 8.2 - 9.4, 20 - 80 mM KCl, 1.0 - 3.0 mM MgCl2, 35 - 45 mM TMAC, 0.05 - 0.15% Triton X-100; Preferably, the buffer includes any one or more of the following: 9.5 - 10.5 mM Tris-HCl, pH 8.2 - 8.6, 20 - 30 mM KCl, 1.0 - 2.0 mM MgCl2, 35 - 45 mM TMAC, 0.05 - 0.15% Triton X-100; 9.5 - 10.5 mM Tris-HCl, pH 8.2 - 8.6, 70 - 80 mM KCl, 1.0 - 2.0 mM MgCl2, 35 - 45 mM TMAC, 0.05 - 0.15% Triton X-100; 9.5 - 10.5 mM Tris-HCl, pH 8.2 - 8.6, 70 - 80 mM KCl, 2.01 - 3.0 mM MgCl2, 35 - 45 mM TMAC, 0.05 - 0.15% Triton X-100; 9.5 - 10.5 mM Tris-HCl, pH 8.61 - 9.0, 20 - 30 mM KCl, 1.0 - 2.0 mM MgCl2, 35 - 45 mM TMAC, 0.05 - 0.15% Triton X-100; or 9.5 - 10.5 mM Tris-HCl, pH 9.01 - 9.4, 20 - 30 mM KCl, 1.0 - 2.0 mM MgCl2, 35 - 45 mM TMAC, 0.05 - 0.15% Triton X-100; More preferably, the buffer solution comprises any one or more of the following: 10 mM Tris-HCl, pH 8.4, 25 mM KCl, 1.5 mM MgCl2, 40 mM TMAC, 0.1% Triton X-100; 10 mM Tris-HCl, pH 8.4, 75 mM KCl, 1.5 mM MgCl2, 40 mM TMAC, 0.1% Triton X-100; 10 mM Tris-HCl, pH 8.4, 75 mM KCl, 2.5 mM MgCl2, 40 mM TMAC, 0.1% Triton X-100; 10 mM Tris-HCl, pH 8.8, 25 mM KCl, 1.5 mM MgCl2, 40 mM TMAC, 0.1% Triton X-100; or 10 mM Tris-HCl, pH 9.2, 25 mM KCl, 1.5 mM MgCl2, 40 mM TMAC, 0.1% Triton X-100.

13. Use of the DNA polymerase according to any one of claims 1 to 4 or the PCR kit according to any one of claims 10 to 12 in PCR amplification, library construction or sequencing.

14. A PCR amplification method, characterized in that, The PCR method comprises: performing PCR amplification by using the DNA polymerase according to any one of claims 1 to 4 or the PCR kit according to any one of claims 10 to 12.

15. The PCR amplification method according to claim 14, wherein The PCR amplification method includes: incorporating modified deoxyribonucleotides using the DNA polymerase during the amplification process; Preferably, the modification includes a 3'-O-blocking modification; Preferably, the 3'-O-blocking modification includes a 3'-O-azidomethyl modification.

16. A library construction kit, characterized in that, The library construction kit includes the DNA polymerase according to any one of claims 1 to 4.

17. The library construction kit according to claim 16, wherein The library construction kit further includes any one or more of the following components: 1) Adapter sequences for library construction; 2) Buffers for providing a library construction environment; 3) Enzymes for library construction, including one or more of a DNA fragmentation enzyme, an end repair enzyme, or a ligase; 4) Modified deoxyribonucleotides.

18. A library construction method, characterized in that, The library construction method includes: performing library construction using the DNA polymerase according to any one of claims 1 to 4 or the library construction kit according to claim 16 or 17.

19. The library construction method according to claim 18, wherein The library construction method includes: Performing PCR amplification on the sequencing fragment linked with a sequencing adapter using the DNA polymerase to obtain a DNA library; Preferably, the library construction method further includes: incorporating modified deoxyribonucleotides using the DNA polymerase during the DNA amplification process.

20. A sequencing kit, characterized in that, The sequencing kit includes the DNA polymerase according to any one of claims 1 to 4.

21. The sequencing kit according to claim 20, characterized in that, The sequencing kit further includes any one or more of the following components: 1) Primers for complementary pairing with adapter sequences; 2) Dideoxynucleotides; 3) dNTPs; 4) Nucleic acid probes; 5) Enzymes for sequencing, including a DNA ligase and / or an endonuclease; 6) Buffers for eluting the nucleic acid probes and / or dNTPs; 7) Modified deoxyribonucleotides.

22. A sequencing method, characterized in that, The sequencing method includes: performing sequencing using the DNA polymerase according to any one of claims 1 to 4 or the sequencing kit according to claim 20 or 21.

23. The sequencing method according to claim 22, characterized in that, The sequencing method includes performing PCR amplification on the sample to be sequenced using the DNA polymerase to obtain a DNA library; Performing sequencing on the DNA library using a sequencing technique to obtain a sequencing result; Preferably, the sequencing method further includes: Incorporating modified deoxyribonucleotides using the DNA polymerase during the PCR amplification process.

Images