Recombinant KOD polymerase

Abstract

Provided is a recombinant KOD polymerase. The recombinant KOD polymerase has a sequence having at least 85% identity with the sequence of a wild-type KOD polymerase, wherein the recombinant KOD polymerase contains one or more mutations at one or more of the following positions corresponding to the sequence of the wild-type KOD polymerase: positions 141, 143, 408, 409, 410, and 485. The recombinant KOD polymerase has DNA polymerase activity, wherein the sequence of the wild-type KOD polymerase is as shown in SEQ ID NO: 1.

Description

Recombinant KOD polymerase Technical Field

[0001] This application relates to the field of molecular cloning technology, specifically to a recombinant KOD polymerase. Background Technology

[0002] DNA polymerases, as key mediators of DNA amplification, play a crucial role in molecular cloning and nucleic acid sequencing. Commercially used DNA polymerases are mostly modified from B-family DNA polymerases derived from archaea, such as KOD (Thermococcus kodakarensis), 9°N (Thermococcus sp. 9oN-7), Tgo (Thermococcus gorgonarius), DTok (Desulfurococcus sp. Tok), Pab (Pyrococcus abyssi), and Deep Vent (Deep Vent DNA Polymerase|Pyrococcus). These modified DNA polymerases possess the ability to polymerize modified nucleotides (i.e., modified nucleotides with reversible blocking groups at the 3' end), and can be used in next-generation sequencing (NGS) technology, such as sequencing-by-synthesis (SBS).

[0003] KOD DNA polymerase is a thermostable DNA polymerase capable of rapidly and accurately replicating DNA. Modified KOD DNA polymerases are widely used in NGS. Developing novel KOD DNA polymerase mutants with improved polymerization activity of modified nucleotides is of great significance for improving the sequencing efficiency and accuracy of NGS.

[0004] Summary of the Invention

[0005] The embodiments of this application provide a recombinant KOD polymerase and its application.

[0006] The first aspect of this application provides a recombinant KOD polymerase having a sequence with at least 85% identity to a wild-type KOD polymerase sequence, wherein the recombinant KOD polymerase contains one or more mutations corresponding to the wild-type KOD polymerase sequence selected from the following sites: positions 141, 143, 408, 409, 410, and 485, wherein the recombinant KOD polymerase has DNA polymerase activity, and wherein the sequence of the wild-type KOD polymerase is shown in SEQ ID NO: 1.

[0007] In some embodiments, the recombinant KOD polymerase contains at least two, at least three, at least four, at least five, or at least six mutations corresponding to the wild-type KOD polymerase sequence selected from the following sites: positions 141, 143, 408, 409, 410, and 485.

[0008] In some embodiments, the mutation is an amino acid substitution, deletion, and / or insertion.

[0009] In some embodiments, the mutation is an amino acid substitution, and the amino acid substitution at the site is independently selected from the following:

[0010] The 141st amino acid is replaced with A;

[0011] The 143rd amino acid is replaced with A;

[0012] The 408th amino acid is replaced with G, A, V, P, N, M, or T;

[0013] The 409th amino acid is replaced with T, A, L, I, V, G, or S;

[0014] The 410th amino acid is replaced with I, D, L, M, G, E, V, Q, or A;

[0015] The 485th amino acid is replaced with L.

[0016] In some embodiments, the recombinant KOD polymerase contains one or more mutations corresponding to the wild-type KOD polymerase sequence selected from positions 408, 409, and 410.

[0017] In some embodiments, the recombinant KOD polymerase comprises a mutation at position 409 and a mutation at position 408 or 410 corresponding to the wild-type KOD polymerase sequence. In some embodiments, the amino acid substitutions at these positions are independently selected from the following:

[0018] The amino acid at position 408 is replaced with A, V, M, N, or G;

[0019] The amino acid at position 409 is replaced with S, G, A, or V;

[0020] The 410th amino acid is replaced with V.

[0021] In some embodiments, the recombinant KOD polymerase has the following mutations: Y409S+P410V+A485L, L408A+Y409G+A485L, L408V+Y409A+A485L, L408M+Y409A+A485L, L408N+Y409A+A485L, Y409G+P410V+A485L, or L408G+Y409V+A485L.

[0022] In some embodiments, the recombinant KOD polymerase comprises mutations at the following three sites corresponding to the wild-type KOD polymerase sequence: positions 408, 409, and 410, wherein amino acid position 408 is replaced with G, A, V, P, N, M, or T; amino acid position 409 is replaced with T, A, L, I, V, G, or S; and amino acid position 410 is replaced with I, D, L, M, G, E, V, Q, or A.

[0023] In some embodiments, the recombinant KOD polymerase has the following mutations: L408G+Y409T+P410I, L408G+Y409A+P410D, L408G+Y409L+P410L, L408G+Y409A+P410M, L408G+Y409I+P410G, L408A+Y409V+P410E, L408A+Y409V+P410M, L408G+Y409T+P410G, L408V+Y409T+P410G, L408P +Y409V+P410L, L408A+Y409I+P410L, L408G+Y409V+P410G, L408G+Y409V+P410M, L408G+Y409V+P410V, L408A+Y409V+P410L, L408A+Y409A+P410Q, L408A+Y409V+P410A, L408N+Y409A+P410D, L408V+Y409V+P410V, or L408T+Y409V+P410L.

[0024] In some embodiments, the recombinant KOD polymerase contains a mutation corresponding to position 141 and / or 143 of the wild-type KOD polymerase sequence, the mutation being optionally D141A and / or D143A.

[0025] In some embodiments, the recombinant KOD polymerase comprises mutations at the following three sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, and 485. In some embodiments, the mutation is D141A-D143A-A485L, and the sequence of the recombinant KOD polymerase is shown in SEQ ID NO: 2.

[0026] In some embodiments, the recombinant KOD polymerase contains mutations at the following five sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, 408, 409, and 410. In some embodiments, the recombinant KOD polymerase has the following mutations:

[0027] D141A+D143A+Y409S+P410V+A485L、

[0028] D141A+D143A+L408A+Y409G+A485L、

[0029] D141A+D143A+L408V+Y409A+A485L、

[0030] D141A+D143A+L408M+Y409A+A485L、

[0031] D141A+D143A+L408N+Y409A+A485L、

[0032] D141A+D143A+Y409G+P410V+A485L or

[0033] D141A+D143A+L408G+Y409V+A485L.

[0034] In some embodiments, the recombinant KOD polymerase comprises mutations at the following six sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, 408, 409, 410, and 485, wherein the mutations at these sites are independently selected from the following: amino acid substitution at position 141 for A; amino acid substitution at position 143 for A; amino acid substitution at position 408 for G, A, V, or M; amino acid substitution at position 409 for A, I, V, G, or S; amino acid substitution at position 410 for L, G, V, Q, or A; and amino acid substitution at position 485 for L.

[0035] In some embodiments, the recombinant KOD polymerase has the following mutations: D141A+D143A+L408A+Y409A+P410Q+A485L, D141A+D143A+L408G+Y409V+P410V+A485L, D141A+D143A+L408A+Y409I+P410L+A485L, D141A+D143A+L408G+Y409I+P410G+A485L, D141A+D143A+L408G+Y409V+P410G+A485L, or D141A+D143A+L408A+Y409V+P410A+A485L.

[0036] In some embodiments, the recombinant KOD polymerase has the following mutations: D141A+D143A+L408A+Y409A+P410Q+A485L, D141A+D143A+L408G+Y409V+P410V+A485L, D141A+D143A+L408A+Y409I+P410L+A485L, D141A+D143A+L408G+Y409I+P410G+A485L, or D141A+D143A+L408G+Y409V+P410G+A485L.

[0037] The second aspect of this application provides a polynucleotide that encodes a recombinant KOD polymerase or its complementary sequence as described in any one of the first aspects of this application.

[0038] A third aspect of this application provides a vector comprising a polynucleotide as described in any one of the second aspect embodiments of this application.

[0039] The fourth aspect of this application provides a cell comprising a polynucleotide as described in any one of the second aspect of this application or a vector as described in any one of the third aspect of this application, or expressing a recombinant KOD polymerase as described in any one of the first aspect of this application.

[0040] The fifth aspect of this application provides a kit comprising a recombinant KOD polymerase as described in any one of the first aspect of this application, a polynucleotide as described in any one of the second aspect of this application, a vector as described in any one of the third aspect of this application, and / or cells as described in any one of the fifth aspect of this application.

[0041] The sixth aspect of this application provides for the use of the recombinant KOD polymerase as described in any one of the first aspect embodiments of this application in molecular cloning, molecular quantitative analysis, molecular marker detection and / or sequencing.

[0042] The seventh aspect of this application provides for the use of the recombinant KOD polymerase as described in any one of the first aspect embodiments of this application in incorporating nucleotides and / or nucleotide analogs into molecular sequences or in preparing nucleotide and / or nucleotide analog polymers, wherein the nucleotide analogs are selected from one or two of the following: modified nucleotides and non-natural nucleotides.

[0043] In some embodiments, the modification is a reversible blocking group modification, which may optionally occur at the 3' sugar ring hydroxyl group and / or base.

[0044] In some embodiments, the non-natural nucleotide is a non-natural nucleotide aptamer.

[0045] An eighth aspect of this application provides a method for incorporating nucleotides and / or nucleotide analogs into a nucleic acid sequence or preparing a polymer of nucleotides and / or nucleotide analogs, comprising: contacting a template nucleic acid sequence, nucleotides and / or nucleotide analogs with a recombinant KOD polymerase as described in any one of the first aspects of this application; and, under the catalysis of the recombinant KOD polymerase, attaching the nucleotides and / or nucleotide analogs to the complementary strand of the template nucleic acid sequence to incorporate the nucleotides and / or nucleotide analogs into the nucleic acid sequence or to prepare the polymer of nucleotides and / or nucleotide analogs.

[0046] In some embodiments, the modification is a reversible blocking group modification, which may optionally occur at the 3' sugar ring hydroxyl group and / or base. In some embodiments, the non-natural nucleotide is a non-natural nucleotide aptamer.

[0047] A ninth aspect of this application provides a nucleic acid sequencing method, comprising: contacting a nucleic acid to be tested, sequencing primers, nucleotides and / or nucleotide analogs with a recombinant KOD polymerase as described in any one of the first aspects of this application; and, under the catalysis of the recombinant KOD polymerase, adding the nucleotides and / or nucleotide analogs to the 3' end of the sequencing primers that are bound to the nucleic acid to be tested, and emitting a detectable signal to achieve the nucleic acid sequencing.

[0048] The technical solution of this application achieves the following technical effects:

[0049] The recombinant KOD polymerase proposed in this application has a stronger nucleotide polymerization ability than its wild type, especially for modified nucleotides (e.g., nucleotides containing blocking groups). It exhibits higher incorporation efficiency and stronger catalytic activity, which can be used for molecular cloning and sequencing (especially NGS), and can effectively improve sequencing efficiency and sequencing accuracy. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 is a gel electrophoresis diagram of the recombinant KOD polymerase according to an embodiment of this application;

[0052] Figure 2 is a gel electrophoresis diagram of the recombinant KOD polymerase according to an embodiment of this application. Detailed Implementation

[0053] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0054] This application is based on the inventor's following understanding:

[0055] KOD DNA polymerase belongs to the B family of DNA polymerases. Modified KOD DNA polymerases possess thermostable properties and can incorporate native nucleotides or nucleotide analogs into molecular sequences. Modified KOD DNA polymerases have a wide range of applications, such as molecular sequencing, particularly NGS characterized by sequencing-by-synthesis. NGS uses nucleotides with a modifying group at the 3' sugar hydroxyl group, thereby blocking the addition of the next nucleotide. Using nucleotides, for example, with a 3'-O-blocking group allows for controlled incorporation of nucleotides into polynucleotide chains. After each nucleotide is added, the presence of the 3'-O-blocking group prevents other nucleotides from being added to the chain. Removing the blocking group restores the native free 3' hydroxyl group, allowing the addition of the next nucleotide. This reversible blocking enables the resolution of individual single bases in sequencing.

[0056] Based on this, the inventors, through numerous experiments and tests, developed a variety of recombinant KOD polymerases, which, compared to the wild-type KOD polymerase sequence (as shown in SEQ ID NO: 1), exhibit mutations at one or more sites. The recombinant KOD polymerases proposed in this application have DNA polymerase activity and are capable of incorporating modified nucleotides or non-natural nucleotides into the molecular sequence, demonstrating enhanced catalytic activity and incorporation rate.

[0057] In this embodiment, "incorporation" refers to linking the nucleotide to the free 3' hydroxyl group of a second nucleotide by forming a phosphodiester bond with the 5' phosphate group of the nucleotide. The second nucleotide to which this nucleotide is linked typically appears at the 3' end of the polynucleotide chain, and this nucleotide can be a natural nucleotide dNTP, a modified nucleotide, or a non-natural nucleotide.

[0058] In this application, “improved / enhanced / enhanced incorporation” refers to an increase in the incorporation efficiency and / or observed incorporation rate of the recombinant KOD polymerase for at least one nucleotide, particularly a modified nucleotide, compared to the control polymerase. However, this application is not limited to improvements in the absolute incorporation rate of modified nucleotides. As explained below, the recombinant KOD polymerase can also incorporate other types of modified nucleotides and so-called dark nucleotides or non-natural nucleotides (such as non-natural nucleotide aptamers, etc.). Therefore, “improved / enhanced / enhanced incorporation” should be interpreted accordingly to also cover any improvements in these other properties, regardless of whether the incorporation rate is increased. The “improvement” need not be constant across all cycles. Therefore, “improvement” can be the ability to incorporate the nucleotide and / or nucleic acid analogue at lower temperatures and / or over a wider temperature range than the control enzyme. In this document, “improvement” can be the ability to incorporate nucleotides and / or nucleic acid analogues when using lower concentrations of modified nucleotides as substrates.

[0059] In this application, "nucleotide" can refer to naturally occurring nucleotides, modified nucleotides, and xeno-nucleic acid (XNA), such as those disclosed in WO2022 / 083686A1. In some embodiments, the modified nucleotide can be a nucleotide modified with a reversible blocking group, wherein the reversible blocking group modification can occur at the 3' sugar ring hydroxyl group and / or base, and the reversible blocking group can be selected from one or more of the following: alkyl, aralkyl, alkenyl, alkynyl, allyl (e.g., 3'-O-allyl), aryl, heteroaryl, heterocyclic, benzyl, azide group, azide group (e.g., 3'-O-azidomethyl), amino, ketone, isocyanate group, phosphate ester group, carbonate group, thio, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amide group, etc., which dissociate under aqueous conditions to produce a molecule with a free 3'-OH. In this application, the xeno-nucleic acid can be a xeno-nucleic acid aptamer.

[0060] In this embodiment, the nucleotides may also bear markers to facilitate their detection. Preferably, the markers are fluorescent markers. Each type of nucleotide may bear different fluorescent markers. However, the detectable markers are not necessarily fluorescent markers. Any marker that allows the detection of nucleotide incorporation in the DNA sequence can be used, and this application does not limit the specific type of marker.

[0061] In the embodiments of this application, recombinant KOD polymerase refers to a polymerase that has at least one amino acid mutation compared to wild-type KOD polymerase, where "mutation" can refer to substitution. In some cases, these mutations are conserved mutations to maintain the overall charge distribution of the protein. However, this application is not limited to conserved substitutions. The recombinant KOD polymerase proposed in the embodiments of this application may also contain non-conserved substitutions. Furthermore, the mutation in the recombinant KOD polymerase proposed in the embodiments of this application can also be the deletion or addition of one or more amino acids to the protein, as long as the recombinant KOD polymerase still has DNA polymerase activity and has improved incorporation efficiency / rate compared to the control polymerase.

[0062] In this application embodiment, the term "percentage of identity" for nucleic acid or polypeptide sequences is defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to a known polypeptide after arranging the sequence to obtain the maximum percentage of identity and introducing gaps (if necessary) to achieve the maximum percentage of homology. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using algorithms employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA. 87, 2264-2268), programs tailored for sequence similarity searches.

[0063] In the embodiments of this application, the recombinant KOD polymerase has a sequence (including endpoint values) that is at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to the wild-type KOD polymerase sequence (SEQ ID NO: 1), wherein the percentage of identity generally describes the degree to which the two sequences are the same, i.e., it generally describes the percentage of nucleotides that correspond to the same nucleotides in the reference sequence at their sequence positions. In some embodiments, the recombinant KOD polymerase has at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, or at least 99%, at least 99.1% compared to SEQ ID NO: 1. An amino acid sequence with at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or any value between these values ​​(e.g., sequence identity represented by an infinite number of decimals between two adjacent integers), but less than 100% identical, wherein the recombinant KOD polymerase has one or more amino acid mutations compared to SEQ ID NO: 1.

[0064] In the embodiments of this application, the recombinant KOD polymerase has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid mutations compared to SEQ ID NO: 1. In some embodiments, unless otherwise specified, the remaining amino acids of the recombinant KOD polymerase are the same as those in SEQ ID NO: 1 or have conserved substitutions corresponding to the amino acids in SEQ ID NO: 1, except for the indicated mutations.

[0065] In some embodiments, the recombinant KOD polymerase comprises one or more mutations corresponding to the wild-type KOD polymerase sequence (SEQ ID NO: 1) selected from the following sites: positions 141, 143, 408, 409, 410, and 485, and the recombinant KOD polymerase has DNA polymerase activity. The recombinant KOD polymerase with the above-mentioned site mutations proposed in this application exhibits improved catalytic activity and superior efficiency in incorporating modified nucleotides compared to the wild-type KOD polymerase.

[0066] In some embodiments, the recombinant KOD polymerase has an amino acid mutation at position 141 of SEQ ID NO: 1. In some embodiments, the mutation at position 141 of the recombinant KOD polymerase can be D141A.

[0067] In some embodiments, the recombinant KOD polymerase has an amino acid mutation at position 143 of SEQ ID NO: 1. In some embodiments, the mutation at position 143 of the recombinant KOD polymerase can be D143A.

[0068] In some embodiments, the recombinant KOD polymerase has an amino acid mutation at position 408 of SEQ ID NO: 1. In some embodiments, the mutation at position 408 of the recombinant KOD polymerase can be L408G, L408A, L408V, L408P, L408N, L408M, or L408T.

[0069] In some embodiments, the recombinant KOD polymerase has an amino acid mutation at position 409 of SEQ ID NO: 1. In some embodiments, the mutation at position 409 of the recombinant KOD polymerase can be Y409T, Y409A, Y409L, Y409I, Y409V, Y409G, or Y409S.

[0070] In some embodiments, the recombinant KOD polymerase has an amino acid mutation at position 410 of SEQ ID NO: 1. In some embodiments, the mutation at position 410 of the recombinant KOD polymerase can be P410I, P410D, P410L, P410M, P410G, P410E, P410V, P410Q, or P410A.

[0071] In some embodiments, the recombinant KOD polymerase has an amino acid mutation at position 485 of SEQ ID NO: 1. In some embodiments, the mutation at position 485 of the recombinant KOD polymerase can be A485L.

[0072] The recombinant KOD polymerase proposed in this application embodiment may also have any combination of the above-mentioned mutation sites, such as mutations at at least two, at least three, at least four, at least five, or at least six sites from positions 141, 143, 408, 409, 410, and 485, wherein the mutation form at each site may optionally be the substitution form described in the above embodiment.

[0073] In some embodiments, the recombinant KOD polymerase comprises a combination of mutations corresponding to positions 408, 409, and 410 of the wild-type KOD polymerase sequence, wherein the mutation at each position can optionally be a substitution as described in the above embodiments, for example, L408G+Y409T+P410I; L408G-Y409T-P410D; L408G-Y409T-P410L; L408G-Y409T-P410M; L408G-Y409T-P410G; L408G-Y409T-P410E; L408G-Y409T-P410V; L408G-Y409T-P410Q; L408G-Y409T-P410 A; L408G-Y409A-P410I; L408G-Y409A-P410D; L408G-Y409A-P410L; L408G -Y409A-P410M; L408G-Y409A-P410G; L408G-Y409A-P410E; L408G-Y409A-P 410V; L408G-Y409A-P410Q; L408G-Y409A-P410A; L408G-Y409L-P410I; L40 8G-Y409L-P410D; L408G-Y409L-P410L; L408G-Y409L-P410M; L408G-Y409L -P410G; L408G-Y409L-P410E; L408G-Y409L-P410V; L408G-Y409L-P410Q; L408G-Y409L-P410A; L408G-Y409I-P410I; L408G-Y409I-P410D; L408G-Y4 09I-P410L; L408G-Y409I-P410M; L408G-Y409I-P410G; L408G-Y409I-P41 0E;L408G-Y409I-P410V;L408G-Y409I-P410Q;L408G-Y409I-P410A;L408G -Y409V-P410I; L408G-Y409V-P410D; L408G-Y409V-P410L; L408G-Y409V- P410M; L408G-Y409V-P410G; L408G-Y409V-P410E; L408G-Y409V-P410V; L4 08G-Y409V-P410Q; L408G-Y409V-P410A; L408G-Y409G-P410I; L408G-Y409 G-P410D; L408G-Y409G-P410L; L408G-Y409G-P410M; L408G-Y409G-P410G;L408G-Y409G-P410E;L408G-Y409G-P410V;L408G-Y409G-P410Q;L408G-Y409G-P410A;L408G-Y409S-P410I;L408G-Y409S-P410D;L408G-Y409S-P410L;L408G-Y409S-P410M;L408G-Y409S-P410G;L408G-Y409S-P410E;L408G-Y409S-P410V;L408G-Y409S-P410Q;L408G-Y409S-P410A;L408A-Y409T-P410I;L408A-Y409T-P410D;L408A-Y409T-P410L;L408A-Y409T-P410M;L408A-Y409T-P410G;L408A-Y409T-P410E;L408A-Y409T-P410V;L408A-Y409T-P410Q;L408A-Y409T-P410A;L408A-Y409A-P410I;L408A-Y409A-P410D;L408A-Y409A-P410L;L408A-Y409A-P410M;L408A-Y409A-P410G;L408A-Y409A-P410E;L408A-Y409A-P410V;L408A-Y409A-P410Q;L408A-Y409A-P410A;L408A-Y409L-P410I;L408A-Y409L-P410D;L408A-Y409L-P410L;L408A-Y409L-P410M;L408A-Y409L-P410G;L408A-Y409L-P410E;L408A-Y409L-P410V;L408A-Y409L-P410Q;L408A-Y409L-P410A;L408A-Y409I-P410I;L408A-Y409I-P410D;L408A-Y409I-P410L;L408A-Y409I-P410M;L408A-Y409I-P410G;L408A-Y409I-P410E;L408A-Y409I-P410V;L408A-Y409I-P410Q;L408A-Y409I-P410A;L408A-Y409V-P410I;L408A-Y409V-P410D;L408A-Y409V-P410L;L408A-Y409V-P410M;L408A-Y409V-P410G;L408A-Y409V-P410E;L408A-Y409V-P410V;L408A-Y409V-P410Q;L408A-Y409V-P410A;L408A-Y409G-P410I;L408A-Y409G-P410D;L408A-Y409G-P410L;L408A-Y409G-P410M;L408A-Y409G-P410G;L408A-Y409G-P410E;L408A-Y409G-P410V;L408A-Y409G-P410Q;L408A-Y409G-P410A;L408A-Y409S-P410I;L408A-Y409S-P410D;L408A-Y409S-P410L;L408A-Y409S-P410M;L408A-Y409S-P410G;L408A-Y409S-P410E;L408A-Y409S-P410V;L408A-Y409S-P410Q;L408A-Y409S-P410A;L408V-Y409T-P410I;L408V-Y409T-P410D;L408V-Y409T-P410L;L408V-Y409T-P410M;L408V-Y409T-P410G;L408V-Y409T-P410E;L408V-Y409T-P410V;L408V-Y409T-P410Q;L408V-Y409T-P410A;L408V-Y409A-P410I;L408V-Y409A-P410D;L408V-Y409A-P410L;L408V-Y409A-P410M;L408V-Y409A-P410G;L408V-Y409A-P410E;L408V-Y409A-P410V;L408V-Y409A-P410Q;L408V-Y409A-P410A;L408V-Y409L-P410I;L408V-Y409L-P410D;L408V-Y409L-P410L;L408V-Y409L-P410M;L408V-Y409L-P410G;L408V-Y409L-P410E;L408V-Y409L-P410V;L408V-Y409L-P410Q;L408V-Y409L-P410A;L408V-Y409I-P410I; L408V-Y409I-P410D;L408V-Y409I-P410L;L408V-Y409I-P410M;L408V-Y409I-P410G;L408V-Y409I-P410E;L408V-Y409I-P410V;L408V-Y409I-P410Q;L408V-Y409I-P410A;L408V-Y409V-P410I;L408V-Y409V-P410D;L408V-Y409V-P410L;L408V-Y409V-P410M;L408V-Y409V-P410G;L408V-Y409V-P410E;L408V-Y409V-P410V;L408V-Y409V-P410Q;L408V-Y409V-P410A;L408V-Y409G-P410I;L408V-Y409G-P410D;L408V-Y409G-P410L;L408V-Y409G-P410M;L408V-Y409G-P410G;L408V-Y409G-P410E;L408V-Y409G-P410V;L408V-Y409G-P410Q;L408V-Y409G-P410A;L408V-Y409S-P410I;L408V-Y409S-P410D;L408V-Y409S-P410L;L408V-Y409S-P410M;L408V-Y409S-P410G;L408V-Y409S-P410E;L408V-Y409S-P410V;L408V-Y409S-P410Q;L408V-Y409S-P410A;L408P-Y409T-P410I;L408P-Y409T-P410D;L408P-Y409T-P410L;L408P-Y409T-P410M;L408P-Y409T-P410G;L408P-Y409T-P410E;L408P-Y409T-P410V;L408P-Y409T-P410Q;L408P-Y409T-P410A;L408P-Y409A-P410I;L408P-Y409A-P410D;408P-Y409A-P410L;L408P-Y409A-P410M;L408P-Y409A-P410G;L408P-Y409A-P410E;L408P-Y409A-P410V;L408P-Y409A-P410Q;L408P-Y409A-P410A;L408P-Y409L-P410I;L408P-Y409L-P410D;L408P-Y409L-P410L;L408P-Y409L-P410M;L408P-Y409L-P410G;L408P-Y409L-P410E;L408P-Y409L-P410V;L408P-Y409L-P410Q;L408P-Y409L-P410A;L408P-Y409I-P410I;L408P-Y409I-P410D;L408P-Y409I-P410L;L408P-Y409I-P410M;L408P-Y409I-P410G;L408P-Y409I-P410E;L408P-Y409I-P410V;L408P-Y409I-P410Q;L408P-Y409I-P410A;L408P-Y409V-P410I;L408P-Y409V-P410D;L408P-Y409V-P410L;L408P-Y409V-P410M;L408P-Y409V-P410G;L408P-Y409V-P410E;L408P-Y409V-P410V;L408P-Y409V-P410Q;L408P-Y409V-P410A;L408P-Y409G-P410I;L408P-Y409G-P410D;L408P-Y409G-P410L;L408P-Y409G-P410M;L408P-Y409G-P410G;L408P-Y409G-P410E;L408P-Y409G-P410V;L408P-Y409G-P410Q;L408P-Y409G-P410A;L408P-Y409S-P410I;L408P-Y409S-P410D;L408P-Y409S-P410L;L408P-Y409S-P410M;L408P-Y409S-P410G;L408P-Y409S-P410E;L408P-Y409S-P410V;L408P-Y409S-P410Q;L408P-Y409S-P410A;L408N-Y409T-P410I;L408N-Y409T-P410D;L408N-Y409T-P410L;L408N-Y409T-P410M;L408N-Y409T-P410G;L408N-Y409T-P410E;L408N-Y409T-P410V;L408N-Y409T-P410Q;L408N-Y409T-P410A;L408N-Y409A-P410I;L408N-Y409A-P410D;L408N-Y409A-P410L;L408N-Y409A-P410M;L408N-Y409A-P410G;L408N-Y409A-P410E;L408N-Y409A-P410V;L408N-Y409A-P410Q;L408N-Y409A-P410A;L408N-Y409L-P410I;L408N-Y409L-P410D;L408N-Y409L-P410L;L408N-Y409L-P410M;L408N-Y409L-P410G;L408N-Y409L-P410E;L408N-Y409L-P410V;L408N-Y409L-P410Q;L408N-Y409L-P410A;L408N-Y409I-P410I;L408N-Y409I-P410D;L408N-Y409I-P410L;L408N-Y409I-P410M;L408N-Y409I-P410G;L408N-Y409I-P410E;L408N-Y409I-P410V;L408N-Y409I-P410Q;L408N-Y409I-P410A;L408N-Y409V-P410I;L408N-Y409V-P410D;L408N-Y409V-P410L;L408N-Y409V-P410M;L408N-Y409V-P410G;L408N-Y409V-P410E;L408N-Y409V-P410V;L408N-Y409V-P410Q;L408N-Y409V-P410A;L408N-Y409G-P410I;L408N-Y409G-P410D;L408N-Y409G-P410L;L408N-Y409G-P410M;L408N-Y409G-P410G;L408N-Y409G-P410E;L408N-Y409G-P410V;L408N-Y409G-P410Q;L408N-Y409G-P410A;L408N-Y409S-P410I;L408N-Y409S-P410D;L408N-Y409S-P410L;L408N-Y409S-P410M;L408N-Y409S-P410G;L408N-Y409S-P410E;L408N-Y409S- P410V;L408N-Y409S-P410Q;L408N-Y409S-P410A;L408M-Y409T-P410I;L408M-Y409T-P410D;L408M-Y409T-P410L;L408M-Y409T-P410M;L408M-Y409T-P410G;L408M-Y409T-P410E;L408M-Y409T-P410V;L408M-Y409T-P410Q;L408M-Y409T-P410A;L408M-Y409A-P410I;L408M-Y409A-P410D;L408M-Y409A-P410L;L408M-Y409A-P410M;L408M-Y409A-P410G;L408M-Y409A-P410E;L408M-Y409A-P410V;L408M-Y409A-P410Q;L408M-Y409A-P410A;L408M-Y409L-P410I;L408M-Y409L-P410D;L408M-Y409L-P410L;L408M-Y409L-P410M;L408M-Y409L-P410G;L408M-Y409L-P410E;L408M-Y409L-P410V;L408M-Y409L-P410Q;L408M-Y409L-P410A;L408M-Y409I-P410I;L408M-Y409I-P410D;L408M-Y409I-P410L;L408M-Y409I-P410M;L408M-Y409I-P410G;L408M-Y409I-P410E;L408M-Y409I-P410V;L408M-Y409I-P410Q;L408M-Y409I-P410A;L408M-Y409V-P410I;L408M-Y409V-P410D;L408M-Y409V-P410L;L408M-Y409V-P410M;L408M-Y409V-P410G;L408M-Y409V-P410E;L408M-Y409V-P410V;L408M-Y409V-P410Q;L408M-Y409V-P410A;L408M-Y409G-P410I;L408M-Y409G-P410D;L408M-Y409G-P410L;L408M-Y409G-P410M;L408M-Y409G-P410G;L408M-Y409G-P410E;L408M-Y409G-P410V;L408M-Y409G-P410Q;L408M-Y409G-P410A;L408M-Y409S-P410I;L408M-Y409S-P410D;L408M-Y409S-P410L;L408M-Y409S-P410M;L408M-Y409S-P410G;L408M-Y409S-P410E;L408M-Y409S-P410V;L408M-Y409S-P410Q;L408M-Y409S-P410A;L408T-Y409T-P410I;L408T-Y409T-P410D;L408T-Y409T-P410L;L408T-Y409T-P410M;L408T-Y409T-P410G;L408T-Y409T-P410E;L408T-Y409T-P410V;L408T-Y409T-P410Q;L408T-Y409T-P410A;L408T-Y409A-P410I;L408T-Y409A-P410D;L408T-Y409A-P410L;L408T-Y409A-P410M;L408T-Y409A-P410G;L408T-Y409A-P410E;L408T-Y409A-P410V;L408T-Y409A-P410Q;L408T-Y409A-P410A;L408T-Y409L-P410I;L408T-Y409L-P410D;L408T-Y409L-P410L;L408T-Y409L-P410M;L408T-Y409L-P410G;L408T-Y409L-P410E;L408T-Y409L-P410V;L408T-Y409L-P410Q;L408T-Y409L-P410A;L408T-Y409I-P410I;L408T-Y409I-P410D;L408T-Y409I-P410L;L408T-Y409I-P410M;L408T-Y409I-P410G;L408T-Y409I-P410E;L408T-Y409I-P410V;L408T-Y409I-P410Q;L408T-Y409I-P410A;L408T-Y409V-P410I;L408T-Y409V-P410D;L408T-Y409V-P410L;L408T-Y409V-P410M;L408T-Y409V-P410G;L408T-Y409V-P410E;L408T-Y409V-P410V;L408T-Y409V-P410Q;L408T-Y409V-P410A;L408T-Y409G-P410I;L408T-Y409G-P410D;L408T-Y409G-P410L;L408T-Y409G-P410M;L408T-Y409G-P410G;L408T-Y409G-P410E;L408T-Y409G-P410V;L408T-Y409G-P410Q;L408T-Y409G-P410A;L408T-Y409S-P410I;L408T-Y409S-P410D;L408T-Y409S-P410L;L408T-Y409S-P410M; L408T-Y409S-P410G; L408T-Y409S-P410E; L408T-Y409S-P410V; L408T-Y409S-P410Q or L408T-Y409S-P410A, preferably Y409S+P410V+A485L or L408A+Y40 9G+A485L, L408V+Y409A+A485L, L408M+Y409A+A485L, L408N+Y409A+A485L, Y409G+ P410V+A485L, L408G+Y409V+A485L, L408G+Y409T+P410I, L408G+Y409A+P410D, L408 G+Y409L+P410L, L408G+Y409A+P410M, L408G+Y409I+P410G, L408A+Y409V+P410E, L 408A+Y409V+P410M, L408G+Y409T+P410G, L408V+Y409T+P410G, L408P+Y409V+P410 L, L408A+Y409I+P410L, L408G+Y409V+P410G, L408G+Y409V+P410M, L408G+Y409V+P410V, L408A+Y409V+P410L, L408A+Y409A+P410Q, L408A+Y409V+P410A, L408N+Y409A+P410D, L408V+Y409V+P410V, or L408T+Y409V+P410L.

[0074] In some embodiments, the recombinant KOD polymerase contains mutations (i.e., a combination of two mutations) corresponding to positions 141 and 143 of the wild-type KOD polymerase sequence, wherein the mutations are optionally D141A and D143A.

[0075] In some embodiments, the recombinant KOD polymerase contains mutations at the following three sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, and 485 (i.e., a three-mutation combination), wherein the mutations are optionally D141A-D141A-A485L, and the corresponding recombinant KOD polymerase sequence is shown in SEQ ID NO: 2.

[0076] In some embodiments, the recombinant KOD polymerase further comprises one or two mutations corresponding to the wild-type KOD polymerase sequence selected from the following sites: positions 408, 409, and 410 (i.e., a four-mutation combination or a five-mutation combination), wherein the mutation at each site may optionally be a substitution form as described in the above embodiments.

[0077] In some embodiments, the five mutant combinations of recombinant KOD polymerase can occur at positions 141, 143, 485, 409, and 408 or 410. In some embodiments, the amino acid substitutions at each site are independently selected from the following:

[0078] The amino acid at position 408 is replaced with A, V, M, N, or G;

[0079] The amino acid at position 409 is replaced with S, G, A, or V;

[0080] The 410th amino acid is replaced with V.

[0081] In some specific embodiments, the five-mutation combination of recombinant KOD polymerase can have the following mutation forms:

[0082] D141A+D143A+Y409S+P410V+A485L、

[0083] D141A+D143A+L408A+Y409G+A485L、

[0084] D141A+D143A+L408V+Y409A+A485L、

[0085] D141A+D143A+L408M+Y409A+A485L、

[0086] D141A+D143A+L408N+Y409A+A485L、

[0087] D141A+D143A+Y409G+P410V+A485L or

[0088] D141A+D143A+L408G+Y409V+A485L.

[0089] In some embodiments, the recombinant KOD polymerase may further comprise mutations at the following six sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, 408, 409, 410, and 485 (i.e., a six-mutation combination). In some embodiments, the six-mutation combination of the recombinant KOD polymerase may have the following mutation forms:

[0090] D141A+D143A+L408G+Y409T+P410I+A485L、

[0091] D141A+D143A+L408G+Y409A+P410D+A485L、

[0092] D141A+D143A+L408G+Y409L+P410L+A485L、

[0093] D141A+D143A+L408G+Y409A+P410M+A485L、

[0094] D141A+D143A+L408G+Y409I+P410G+A485L、

[0095] D141A+D143A+L408A+Y409V+P410E+A485L、

[0096] D141A+D143A+L408A+Y409V+P410M+A485L、

[0097] D141A+D143A+L408G+Y409T+P410G+A485L、

[0098] D141A+D143A+L408V+Y409T+P410G+A485L、

[0099] D141A+D143A+L408P+Y409V+P410L+A485L、

[0100] D141A+D143A+L408A+Y409I+P410L+A485L、

[0101] D141A+D143A+L408G+Y409V+P410G+A485L、

[0102] D141A+D143A+L408G+Y409V+P410M+A485L、

[0103] D141A+D143A+L408G+Y409V+P410V+A485L、

[0104] D141A+D143A+L408A+Y409V+P410L+A485L、

[0105] D141A+D143A+L408A+Y409A+P410Q+A485L、

[0106] D141A+D143A+L408A+Y409V+P410A+A485L、

[0107] D141A+D143A+L408N+Y409A+P410D+A485L、

[0108] D141A+D143A+L408V+Y409V+P410V+A485L or

[0109] D141A+D143A+L408T+Y409V+P410L+A485L.

[0110] In some embodiments, the six-mutation combination of recombinant KOD polymerase can occur at positions 141, 143, 408, 409, 410, and 485. In some embodiments, the amino acid substitutions at each site are independently selected from the following:

[0111] The 141st amino acid is replaced with A;

[0112] The 143rd amino acid is replaced with A;

[0113] The amino acid at position 408 is replaced with G, A, V, or M;

[0114] The 409th amino acid is replaced with A, I, V, G, or S;

[0115] The 410th amino acid is replaced with L, G, V, Q, or A;

[0116] The 485th amino acid is replaced with L.

[0117] In some specific embodiments, the six-mutant combination of recombinant KOD polymerase can have the following mutation forms:

[0118] D141A+D143A+L408A+Y409A+P410Q+A485L、

[0119] D141A+D143A+L408G+Y409V+P410V+A485L,

[0120] D141A+D143A+L408A+Y409I+P410L+A485L、

[0121] D141A+D143A+L408G+Y409I+P410G+A485L、

[0122] D141A+D143A+L408G+Y409V+P410G+A485L or

[0123] D141A+D143A+L408A+Y409V+P410A+A485L.

[0124] The recombinant KOD polymerase proposed in this application has a stronger nucleotide polymerization ability than its wild type, especially for modified nucleotides (e.g., 3'-O-blocking modified nucleotides), exhibiting higher incorporation efficiency and stronger catalytic activity. Therefore, it can be used for molecular cloning and sequencing (especially NGS), and can effectively improve sequencing efficiency and sequencing accuracy.

[0125] It is understood that the recombinant KOD polymerase proposed in this application embodiment may also have other modifications as needed, such as surface modifications like macromolecular modifications, small molecule modifications, cross-linking modifications, and / or immobilization modifications; internal modifications like modifications to non-catalytic groups, catalytic groups, the main chain, cofactors, and peptide chain extension; and / or chemical modifications combined with site-directed mutagenesis. The recombinant KOD polymerase proposed in this application embodiment may also have its primary structure modified as needed, such as by adding a terminal tag. These modifications can be achieved by conventional methods in the art as needed, and they also fall within the protection scope of this application.

[0126] This application also provides a polynucleotide encoding the recombinant KOD polymerase described in any of the above embodiments, a vector containing the polynucleotide, a cell containing the polynucleotide, the vector and / or expressing the recombinant KOD polymerase, and a kit containing the recombinant KOD polymerase, the polynucleotide, the vector and / or the cell.

[0127] This application also proposes the use of the recombinant KOD polymerase described in any of the above embodiments in molecular cloning, molecular quantitative analysis, molecular marker detection and / or sequencing, wherein the sequencing may optionally be NGS.

[0128] This application also proposes the application of the recombinant KOD polymerase described in any of the above embodiments in incorporating nucleotides and / or nucleotide analogs into molecular sequences or in preparing nucleotide and / or nucleotide analog polymers, wherein the nucleotide analogs are selected from one or two of the following: modified nucleotides and non-natural nucleotides. In some embodiments, the modification is a reversible blocking group modification, preferably the reversible blocking group modification optionally occurring at the 3' sugar ring hydroxyl group and / or base; in some embodiments, the non-natural nucleotide is a non-natural nucleotide aptamer.

[0129] This application also proposes a method for incorporating nucleotides and / or nucleotide analogs into a nucleic acid sequence or preparing a polymer of nucleotides and / or nucleotide analogs, comprising: contacting a template nucleic acid sequence, nucleotides and / or nucleotide analogs with a recombinant KOD polymerase as described in any of the above embodiments; and, under the catalysis of the recombinant KOD polymerase, attaching the nucleotides and / or nucleotide analogs to the complementary strand of the template nucleic acid sequence to incorporate the nucleotides and / or nucleotide analogs into the nucleic acid sequence or to prepare the polymer of nucleotides and / or nucleotide analogs.

[0130] This application also proposes a nucleic acid sequencing method, comprising: contacting a nucleic acid to be tested, sequencing primers, nucleotides and / or nucleotide analogs with a recombinant KOD polymerase as described in any of the above embodiments; and, under the catalysis of the recombinant KOD polymerase, adding the nucleotides and / or nucleotide analogs to the 3' end of the sequencing primers that are bound to the nucleic acid to be tested, and emitting a detectable signal to achieve the nucleic acid sequencing. In some embodiments, the nucleotide analogs contain reversible blocking group modifications.

[0131] It should be noted that the above explanations and descriptions of the recombinant KOD polymerase examples are also applicable to the application of the polynucleotides, vectors, cells, kits, and recombinant KOD polymerase in the embodiments of this application, and will not be repeated here.

[0132] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0133] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0134] Example 1

[0135] 1. Construction of a KOD DNA polymerase mutant library

[0136] 1.1 Construction and transformation of plasmids containing KOD DNA polymerase mutants

[0137] Based on the wild-type KOD DNA polymerase sequence (SEQ ID NO: 1), a triple mutant KOD-M containing KOD DNA polymerase was artificially synthesized. Compared with SEQ ID NO: 1, it contains the mutant form of D141A+E143A+A485L, and its amino acid sequence is shown in SEQ ID NO: 2. Based on this sequence, the recombinant plasmid pD441-KOD-M was constructed and transformed into BL21(DE3) competent cells (Tiangen Biotech (Beijing) Co., Ltd., catalog number: CB105-02). The specific transformation steps are as follows: Add 0.5 μL of 50 ng / μL pD441 / KOD-M plasmid to 100 μL of BL21(DE3) competent cells, gently mix and place on ice for 5 minutes; heat shock at 42℃ for 1 minute and place on ice again for 5 minutes; add 400 μL of antibiotic-free LB liquid medium and shake at 220 rpm at 37℃ for 1 hour; take 100 μL of culture and spread it on kanamycin-resistant LB plates and incubate at 37℃ for 12-16 hours.

[0138] KOD DNA polymerase wild-type sequence SEQ ID NO: 1

[0139] KOD DNA polymerase mutant KOD-M sequence (SEQ ID NO: 2):

[0140] 1.2 Protein Expression and Purification of KOD DNA Polymerase Mutant KOD-M

[0141] 1.2.1 Pick 3-6 healthy single colonies from an LB agar plate and inoculate them into 50 mL of LB liquid medium. Incubate at 37°C for 5-7 h until the OD600 reaches 0.6-4.0. Then, inoculate 1% of the bacterial culture into 2 L of LB liquid medium and incubate at 37°C for 2-4 h until the OD600 reaches 0.6-0.8. Pre-cool the original shaker to 25°C. Add IPTG to the medium to a final concentration of 0.5 mM. Place the bacterial culture in a shaker at 25°C and induce expression at 220 rpm for 12-16 h.

[0142] 1.2.2 After induction of expression, all bacterial cultures were centrifuged at 8000g for 10 min to collect the bacterial cells. Subsequently, Ni column affinity solution A was added at a ratio of 1:10 to resuspend the bacterial cells, and the cells were disrupted by sonication in an ice bath environment.

[0143] 1.2.3 Heating treatment: Preheat the water bath to 75-80℃, put the broken bacterial cells into the water bath, shake and mix well, use a thermometer to detect the internal temperature of the bacterial solution. When it reaches 75℃, start timing for 30 minutes. During this period, shake and mix 3 times every 10 minutes to ensure that it is heated evenly.

[0144] 1.2.4 The heat-treated ultrasonically disrupted fluid was centrifuged at 12000 rpm at 4℃ for 60 min. The supernatant was filtered through a 0.22 μM filter membrane as AKTA ( pure TM Sample loading onto the Cytiva purification column of the 25T purification system.

[0145] 1.2.5 The above sample was loaded into a pretreated nickel chromatography column (HisTrap FF, 5 mL column, brand: Cytiva, catalog number: 17528601) at a flow rate of 2.5 mL / min. After loading, the column was washed with Ni column affinity solution A for 20 column volumes (20 CV). Linear elution with Ni column affinity solution B (0-50%) at 20 CV was then performed. Eluted proteins were collected when the UV absorption peak reached 50 mAu, and collection was stopped when the UV absorption peak decreased to 200 mAu.

[0146] 1.2.6 The collected protein elution peak samples were dialyzed overnight at 4°C. After dialysis, the protein concentration was determined and stored in stock solution at -20°C for subsequent functional activity analysis. The enzyme stock concentration was 1 mg / mL.

[0147] The specific components of the buffer solution used in the purification process are shown below:

[0148] Ni column affinity solution A: 34.8 mM K₂HPO₄, 15.2 mM KH₂PO₄, 300 mM NaCl, 20 mM Imidazole, 5% glycerol, pH 7.4

[0149] Ni column affinity solution B: 34.8 mM K₂HPO₄, 15.2 mM KH₂PO₄, 300 mM NaCl, 500 mM Imidazole, 5% glycerol.

[0150] pH 7.4

[0151] Dialysis buffer: 20 mM Tris-HCl, 200 mM KCl, 0.2 mM EDTA, 5% Glycerol, pH 7.4 @ 25℃

[0152] Stock solution: 10 mM Tris-HCl, 100 mM KCl, 0.1 mM EDTA, 50% Glycerol, pH 7.4 @ 25℃

[0153] 1.3 Construction of KOD DNA polymerase mutant vector

[0154] Using the KOD DNA polymerase mutant expression plasmid pD441 / KOD-M from step 1.1 as a template, rapid PCR amplification was performed, and mutations were simultaneously introduced into two or three sites (408, 409, and 410) of KOD, resulting in a total of 27 mutants: KOD-M1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18 / 19 / 20 / 21 / 22 / 23 / 24 / 25 / 26 / 27. Specific mutation information for each mutant is shown in Table 1 below.

[0155] The rapid PCR reaction system consisted of: 5 μL Pfu DNA Polymerase 10X Buffer with MgSO4, 1 μL dNTP Mix (10 mM each), 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 50 ng template DNA, 0.5 μL Pfu DNA Polymerase (3 U / μL, Promega, catalog number: M7741), and nuclease-free water to a total volume of 50 μL. The forward and reverse primers for each mutant are shown in Table 2. The rapid PCR reaction program was set as follows: 95℃ pre-denaturation for 2 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 8 min, 16 cycles; final extension at 72℃ for 5 min, and storage at 4℃.

[0156] Table 1

[0157] Table 2

[0158] 1 μL of DpnI (20 U / μL, NEB, catalog number: R0176) was added to the PCR product and incubated at 37°C for 2 hours to degrade the template. The reaction product was then transformed into *E. coli* DH5α competent cells (Tiangen, catalog number: CB101-02). The transformation procedure was as follows: 10 μL of the reaction product was added to 100 μL of DH5α competent cells, gently swirl to mix, and incubated on ice for 30 minutes; heat-shocked at 42°C for 1 minute, then incubated on ice again for 10 minutes; 400 μL of antibiotic-free LB agar was added, and the mixture was shaken at 37°C for 220 rpm / min for 1 hour. After centrifuging the culture at 3000g for 3 minutes, a portion of the supernatant was discarded, leaving approximately 100 μL. The culture was resuspended, mixed, and plated onto kanamycin-resistant LB agar plates, and incubated at 37°C for 12-16 hours. Several single colonies were picked and cultured overnight at 37°C for small-scale plasmid DNA extraction. The plasmid was sequenced to verify that the mutation site was correctly introduced. Subsequent protein expression and purification were performed using the verified vectors shown in Table 1.

[0159] 1.4 Protein Expression and Purification of KOD DNA Polymerase Mutants M1-M27

[0160] The expression and purification of KOD DNA polymerase mutants KOD-M1 to M27 were performed according to step 1.2 above. The collected protein elution peak samples were analyzed by SDS-PAGE electrophoresis to determine the protein purity. The results are shown in Figure 1 and Figure 2.

[0161] As shown in Figures 1 and 2, the mutant proteins are of the correct size, stable in expression, and at high concentrations, making them suitable for subsequent experiments.

[0162] Example 2

[0163] 1. Test of the polymerization ability of KOD DNA polymerase mutants to block nucleotides with 3' hydroxyl groups

[0164] Using dATP labeled with Cy5 fluorescent dye and modified with a 3'-O-azidomethyl reversible blocking group as a substrate, and double-stranded DNA labeled with Cy3 fluorescent dye as a template / primer (PT-1: Cy3-CGTGTATGCGTAATAGGATCCCGACTCACTATGGACG (SEQ ID NO: 57); PT-2: Cy3-CGTGTATCGTCCATAGTGAGTCGGGATCCTATTACGC (SEQ ID NO: 58), PT-1 and PT-2 with Cy3 dye modified at the 5' end were mixed in equal proportions, incubated at 80℃ for 10 min, and then cooled to room temperature to obtain the template / primer), the reversible blocking modified nucleotide was incorporated into the high-throughput sequencing process. The polymerization activity of KOD-M DNA polymerase and its mutants KOD-M1 to KOD-M27 for the incorporation of modified single nucleotides was detected by detecting the FRET-Cy5 fluorescence signal generated when the substrate polymerizes onto the template strand due to the interaction distance between Cy3 and Cy5 on a microplate reader.

[0165] The detection method is as follows:

[0166] Prepare a 50 μL reaction system as follows: 5 μL 10× reaction buffer, 40 μM dT / C / GTP, 0.1 mg / mL BSA, 1 μM Cy3-DNA double-stranded template, 4 μM Cy5-modified 3'-O-AzidoMethyl-dATP, 0.5 μg KOD DNA polymerase (SEQ ID NO: 1) or KOD-M DNA polymerase (SEQ ID NO: 2) and its mutants (KOD-M1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18 / 19 / 20 / 21 / 22 / 23 / 24 / 25 / 26 / 27), and bring the nuclease-free water to a final volume of 50 μL. The 10× reaction buffer consists of 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, and 20 mM... MgSO4, pH 8.5 @ 25℃. The reaction system was prepared on ice, transferred to a 384-well plate after preparation, and placed in a microplate reader (BioTek Synergy H1, Agilent) for fluorescence signal detection. The reaction temperature was set to 40℃, and fluorescence signals at 530 / 568nm and 630 / 676nm (FRET Cy5) were collected every 60s for a total reaction time of 1 hour. The negative control was replaced with an equal volume of enzyme stock solution for KOD-M DNA polymerase and its mutant. After the reaction, the raw data were exported and the maximum slope, i.e., the ΔFRET-Cy5 fluorescence value per unit time (ΔRFU), was calculated to characterize the polymerase's polymerization activity for the incorporation of modified single nucleotides.

[0167] The experimental results are shown in Table 3 below. As can be seen from Table 3, no FRET Cy5 fluorescence signal was detected in the negative control group. Increased fluorescence signals were detected in the experimental groups of the 27 DNA polymerase mutants (KOD-M1-M27). No signal was detected in the KOD wild-type (KOD_WT, SEQ ID NO: 1), demonstrating that the above-mentioned mutation modification improved the polymerization activity of KOD DNA polymerase for 3'-O-blocking modified nucleotides. This indicates that by performing one or more mutations at positions 141, 143, 408, 409, 410, and 485 of the KOD DNA polymerase, the catalytic activity of the KOD enzyme can be effectively enhanced, especially the incorporation efficiency / rate of modified nucleotides. This provides mutants with great application potential for sequencing.

[0168] Table 3

[0169] Example 3 - Sequencing performance of the KOD DNA polymerase mutant

[0170] Sequencing tests were performed on KOD-M5, KOD-M13, KOD-M14, KOD-M16, and KOD-M18, among the 27 mutants mentioned above, to evaluate the sequencing performance of the KOD DNA polymerase mutants proposed in the embodiments of this application.

[0171] Table 4 shows the sequencing results of KOD-M5, KOD-M13, KOD-M14, and KOD-M16 on an MGISEQ-500 using a single-end 100-cycle (SE100) sequencing of an Escherichia coli genome library (Ecoli.fa) based on 3'-O-reversible blocking modified nucleotides as substrates.

[0172] Table 4

[0173] As shown in Table 4, sequencing signals were generated in library sequencing catalyzed by recombinant KOD enzymes KOD-M5, KOD-M13, KOD-M14, and KOD-M16. Furthermore, the sequencing parameters of the two experimental replicates of the same mutant were similar, demonstrating that the recombinant KOD enzymes proposed in this application can be effectively used for sequencing with 3'-O-reversibly blocking modified nucleotides as substrates, and that the catalytic effect is stable.

[0174] Table 5 shows the sequencing results of mutant KOD-M18 using 3'-O-reversible blocking modified nucleotides as substrates and 150 cycles (PE150) of paired ends on MGISEQ-2000.

[0175] Table 5

[0176] As shown in Table 5, the recombinant KOD enzyme KOD-M18 can produce sequencing signals in sequencing using 3'-O-reversible blocking modified nucleotides as substrates. Furthermore, based on the Q30 value (referring to a base quality value of 30, an error rate of 0.1%, and an accuracy rate of 99.99%), it can reach 84%, indicating that the proposed method in this application still has superior performance in paired-end long-read sequencing. Moreover, it exhibits stable catalytic effect and high sequencing accuracy in sequencing using 3'-O-reversible blocking modified nucleotides as substrates.

[0177] 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.

[0178] 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.

[0179] All embodiments of this application can be executed individually or in combination with other embodiments, and are all considered to be within the scope of protection claimed by this disclosure.

Claims

1. A recombinant KOD polymerase having a sequence with at least 85% identity to a wild-type KOD polymerase sequence, wherein the recombinant KOD polymerase comprises one or more mutations corresponding to the wild-type KOD polymerase sequence selected from positions 141, 143, 408, 409, 410, and 485, the recombinant KOD polymerase having DNA polymerase activity. The sequence of the wild-type KOD polymerase is shown in SEQ ID NO:

1.

2. The recombinant KOD polymerase according to claim 1, wherein the recombinant KOD polymerase comprises at least two, at least three, at least four, at least five, or at least six mutations corresponding to the wild-type KOD polymerase sequence selected from the following sites: positions 141, 143, 408, 409, 410, and 485.

3. The recombinant KOD polymerase according to claim 1 or 2, wherein the mutation is an amino acid substitution, deletion, and / or insertion.

4. The recombinant KOD polymerase according to claim 3, wherein the mutation is an amino acid substitution, and the amino acid substitution at the site is independently selected from the following: The 141st amino acid is replaced with A; The 143rd amino acid is replaced with A; The 408th amino acid is replaced with G, A, V, P, N, M, or T; The 409th amino acid is replaced with T, A, L, I, V, G, or S; The 410th amino acid is replaced with I, D, L, M, G, E, V, Q, or A; The 485th amino acid is replaced with L.

5. The recombinant KOD polymerase according to any one of claims 1 to 4, wherein the recombinant KOD polymerase comprises one or more mutations corresponding to the wild-type KOD polymerase sequence selected from positions 408, 409 and 410.

6. The recombinant KOD polymerase according to any one of claims 1 to 5, wherein the recombinant KOD polymerase comprises a mutation at position 409 and a mutation at position 408 or 410 corresponding to the wild-type KOD polymerase sequence. Alternatively, the amino acid substitutions at the site are independently selected from the following: The amino acid at position 408 is replaced with A, V, M, N, or G; The amino acid at position 409 is replaced with S, G, A, or V; The 410th amino acid is replaced with V. Preferably, the recombinant KOD polymerase has the following mutations: Y409S+P410V+A485L L408A+Y409G+A485L L408V+Y409A+A485L L408M+Y409A+A485L L408N+Y409A+A485L Y409G+P410V+A485L or L408G+Y409V+A485L.

7. The recombinant KOD polymerase according to any one of claims 1 to 6, wherein the recombinant KOD polymerase comprises mutations at the following three sites corresponding to the wild-type KOD polymerase sequence: positions 408, 409, and 410, wherein The 408th amino acid is replaced with G, A, V, P, N, M, or T; The 409th amino acid is replaced with T, A, L, I, V, G, or S; The 410th amino acid is replaced with I, D, L, M, G, E, V, Q, or A. Preferably, the recombinant KOD polymerase has the following mutations: L408G+Y409T+P410I L408G+Y409A+P410D L408G+Y409L+P410L L408G+Y409A+P410M L408G+Y409I+P410G, L408A+Y409V+P410E L408A+Y409V+P410M, L408G+Y409T+P410G, L408V+Y409T+P410G L408P+Y409V+P410L, L408A+Y409I+P410L, L408G+Y409V+P410G, L408G+Y409V+P410M L408G+Y409V+P410V L408A+Y409V+P410L, L408A+Y409A+P410Q L408A+Y409V+P410A L408N+Y409A+P410D L408V+Y409V+P410V or L408T+Y409V+P410L.

8. The recombinant KOD polymerase according to any one of claims 1 to 7, wherein the recombinant KOD polymerase comprises a mutation corresponding to position 141 and / or 143 of the wild-type KOD polymerase sequence, the mutation optionally being D141A and / or D143A.

9. The recombinant KOD polymerase according to any one of claims 1 to 8, wherein the recombinant KOD polymerase comprises mutations at the following three sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, and 485. Optionally, the mutation is D141A-D143A-A485L, and the sequence of the recombinant KOD polymerase is shown in SEQ ID NO:

2.

10. The recombinant KOD polymerase according to any one of claims 1 to 9, wherein the recombinant KOD polymerase comprises mutations at the following five sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, 408, 409, and 410. Optionally, the recombinant KOD polymerase has the following mutations: D141A+D143A+Y409S+P410V+A485L、 D141A+D143A+L408A+Y409G+A485L、 D141A+D143A+L408V+Y409A+A485L、 D141A+D143A+L408M+Y409A+A485L、 D141A+D143A+L408N+Y409A+A485L、 D141A+D143A+Y409G+P410V+A485L or D141A+D143A+L408G+Y409V+A485L.

11. The recombinant KOD polymerase according to any one of claims 1 to 10, wherein the recombinant KOD polymerase comprises mutations at the following six sites corresponding to the wild-type KOD polymerase sequence: positions 141, 143, 408, 409, 410, and 485, wherein the mutations at said sites are independently selected from the following: The 141st amino acid is replaced with A; The 143rd amino acid is replaced with A; The amino acid at position 408 is replaced with G, A, V, or M; The 409th amino acid is replaced with A, I, V, G, or S; The 410th amino acid is replaced with L, G, V, Q, or A; The 485th amino acid is replaced with L.

12. The recombinant KOD polymerase according to claim 11, wherein the recombinant KOD polymerase has the following mutations: D141A+D143A+L408A+Y409A+P410Q+A485L, D141A+D143A+L408G+Y409V+P410V+A485L, D141A+D143A+L408A+Y409I+P410L+A485L, D141A+D143A+L408G+Y409I+P410G+A485L, D141A+D143A+L408G+Y409V+P410G+A485L, or D141A+D143A+L408A+Y409V+P410A+A485L. Preferably, the recombinant KOD polymerase has the following mutations: D141A+D143A+L408A+Y409A+P410Q+A485L、 D141A+D143A+L408G+Y409V+P410V+A485L, D141A+D143A+L408A+Y409I+P410L+A485L、 D141A+D143A+L408G+Y409I+P410G+A485L or D141A+D143A+L408G+Y409V+P410G+A485L.

13. A polynucleotide encoding a recombinant KOD polymerase or its complementary sequence as described in any one of claims 1 to 12.

14. A vector comprising the polynucleotide as described in claim 13.

15. A cell comprising the polynucleotide of claim 13 or the vector of claim 14, or expressing the recombinant KOD polymerase of any one of claims 1 to 12.

16. A kit comprising the recombinant KOD polymerase as described in any one of claims 1 to 12, the polynucleotide as described in claim 13, the vector as described in claim 14, and / or the cells as described in claim 15.

17. Use of the recombinant KOD polymerase as described in any one of claims 1 to 12 in molecular cloning, molecular quantitative analysis, molecular marker detection and / or sequencing.

18. The use of the recombinant KOD polymerase according to any one of claims 1 to 12 in incorporating nucleotides and / or nucleotide analogs into a molecular sequence or in preparing polymers of nucleotides and / or nucleotide analogs, wherein the nucleotide analog is selected from one or two of the following: modified nucleotides and non-natural nucleotides. Optionally, the modification is a reversible blocking group modification, which may optionally occur at the 3' sugar ring hydroxyl group and / or base. Optionally, the non-natural nucleotide is a non-natural nucleotide aptamer.

19. A method for incorporating nucleotides and / or nucleotide analogs into a nucleic acid sequence or for preparing polymers of nucleotides and / or nucleotide analogs, comprising: Contact the template nucleic acid sequence, nucleotide and / or nucleotide analog with the recombinant KOD polymerase as described in any one of claims 1 to 12; and Catalyzed by the recombinant KOD polymerase, the nucleotides and / or nucleotide analogs are attached to the complementary strand of the template nucleic acid sequence to incorporate the nucleotides and / or nucleotide analogs into the nucleic acid sequence or to prepare the polymer of the nucleotides and / or nucleotide analogs.

20. The method of claim 19, wherein the modification is a reversible blocking group modification, the reversible blocking group modification optionally occurring at the 3' sugar ring hydroxyl group and / or base. Optionally, the non-natural nucleotide is a non-natural nucleotide aptamer.

21. A nucleic acid sequencing method, comprising: Contact the nucleic acid to be tested, sequencing primers, nucleotides and / or nucleotide analogs with the recombinant KOD polymerase as described in any one of claims 1 to 12; and Catalyzed by the recombinant KOD polymerase, the nucleotide and / or nucleotide analogue are added to the 3' end of the sequencing primer that binds to the nucleic acid to be tested, and a detectable signal is emitted to achieve the nucleic acid sequencing.

22. The method of claim 21, wherein the nucleotide analog comprises a reversible blocking group modification.

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