DNA polymerase mutant and use thereof

Abstract

Provided are a DNA polymerase mutant and a use thereof. Compared with a wild-type KOD DNA polymerase, the DNA polymerase mutant has amino acid mutations at sites 408 and 409, or at functionally equivalent sites, and amino acid mutations at at least one of the following 23 sites or functionally equivalent sites: site 141, site 143, site 147, site 383, site 384, site 389, site 485, site 584, site 589, site 397, site 424, site 432, site 445, site 523, site 553, site 561, site 564, site 461, site 481, site 605, site 663, site 711, and site 725; an amino acid sequence of the mutant other than the amino acid mutation sites has at least 90% identity with a corresponding amino acid sequence of the wild-type KOD DNA polymerase; and the wild-type KOD DNA polymerase has an amino acid sequence as shown in SEQ ID NO: 2.

Description

DNA polymerase mutants and their applications Technical Field

[0001] The present application relates to the field of biotechnology, and in particular, to DNA polymerase mutants and applications thereof. Background Art

[0002] DNA polymerase is widely used in PCR technology and plays a vital role in life science research and related fields. One of its primary functions is to efficiently and accurately synthesize a DNA sequence complementary to the template in a 5' to 3' direction, using single-stranded DNA as a template and deoxynucleotides (dNTPs) as substrates. Its structure can be broadly divided into the finger, thumb, and palm regions, with the active site typically located in the palm.

[0003] DNA polymerases are classified into seven families: A, B, C, D, X, Y, and RT. DNA polymerases from different families exhibit structural and functional differences. Family B DNA polymerases typically possess high replication and elongation properties, meaning they can continuously polymerize a large number of nucleotides before dissociating from the DNA template.

[0004] In addition, family B DNA polymerases also possess DNA repair functions, including 3'→5' exonuclease activity, which can detect and remove mismatched bases during PCR amplification, reinsert the correct bases, and continue DNA replication. KOD is a representative of family B DNA polymerases, derived from the thermophilic archaeon Thermococcuskodakarensis. Due to its high-temperature resistance, KOD DNA polymerase has been widely used in PCR amplification, gene sequencing, and other fields in recent years. However, the existing KOD DNA polymerases have low polymerization ability during sequencing, which greatly limits the efficiency of sequencing.

[0005] For this reason, KOD DNA polymerase still needs to be improved.

[0006] Summary of the Invention

[0007] This application is completed by the inventor based on the discovery of the following problems and facts:

[0008] To address the short read lengths and slow reaction rates of existing KOD DNA polymerases in sequencing by synthesis (SBS), the inventors engineered active sites related to the thermostable B family polymerases of thermophilic archaea to obtain new DNA polymerase mutants. By improving the polymerization ability of KOD DNA polymerases during sequencing, the inventors further enhanced the sequencing speed and quality of the sequencing by synthesis (SBS) method.

[0009] To this end, in a first aspect of the present application, a DNA polymerase mutant is provided. Compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 24 positions and functionally equivalent positions: position 141, position 143, position 147, 382, ​​position 383, position 384, position 389, 485, 584, 589, 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725; the amino acid sequence of the mutant other than the amino acid mutation sites is the same as that of the wild-type KOD The corresponding amino acid sequence of the DNA polymerase has at least 90% identity; the wild-type KOD DNA polymerase has the amino acid sequence shown in SEQ ID NO: 2. According to the embodiments of the present application, the DNA polymerase mutant can effectively improve the ability to incorporate dNTPs, thereby improving the sequencing speed and sequencing quality of sequencing methods (such as sequencing by synthesis).

[0010] In some examples of the present application, compared to the wild-type KOD DNA polymerase, the DNA polymerase mutant has an amino acid mutation at position 408, position 409, or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 23 positions and functionally equivalent positions: position 141, position 143, position 147, 383, position 384, position 389, 485, 584, 589, 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725; the amino acid sequence of the mutant other than the amino acid mutation positions has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase; the wild-type KOD DNA polymerase has the amino acid sequence of SEQ ID NO: 141. The amino acid sequence shown in NO:2.

[0011] It should be noted that the amino acid positions in the amino acid sequence of the DNA polymerase mutant described herein are referenced to the amino acid positions in SEQ ID NO: 2. For example, if a DNA polymerase mutant has a 408th (point) mutation, it means that the amino acid at position 408 in SEQ ID NO: 2 has mutated.

[0012] It should be noted that the "functionally equivalent sites" used in the present invention include sites where, under specific circumstances, amino acids or nucleotides mutate but the function or properties of the DNA polymerase do not change.

[0013] It should be noted that at least 90% identity includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0014] It should be noted that, in the context of “the amino acid sequence of the mutant other than the amino acid mutation site is at least 90% identical to the corresponding amino acid sequence of the wild-type KOD DNA polymerase” described herein, “the corresponding amino acid sequence of the wild-type KOD DNA polymerase” refers to the amino acid sequence in the wild-type KOD DNA polymerase that corresponds to the amino acid sequence other than the amino acid mutation site in the mutant.

[0015] According to an embodiment of the present application, the above-mentioned DNA polymerase mutant may further include at least one of the following technical features:

[0016] According to an embodiment of the present application, the amino acid sequence of the mutant other than the amino acid mutation site is identical to the corresponding amino acid sequence of the wild-type KOD DNA polymerase.

[0017] According to the embodiments of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has an amino acid mutation at position 408, position 409, or a functionally equivalent site, and an amino acid mutation at at least one site selected from the following nine sites and functionally equivalent sites: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, and position 589. According to some examples of the present application, the relative activities of the sequencers produced by different mutation sites are different, which can provide more options for actual production needs.

[0018] According to the examples of the present application, compared to the wild-type KOD DNA polymerase, the DNA polymerase mutant has an amino acid mutation at position 408, position 409, or a functionally equivalent site, and an amino acid mutation at at least one site selected from the following two sites and functionally equivalent sites: position 147 and position 584. According to the examples of the present application, after extensive screening, the inventors discovered for the first time that mutating the amino acids at positions 147 and 584 can increase the activity of the DNA polymerase.

[0019] According to the embodiments of the present application, the mutation type of the mutation site is:

[0020] (1) D at position 141 mutated to A, (2) E at position 143 mutated to A, (3) H at position 147 mutated to E, (4) S at position 383 mutated to T, (5) Y at position 384 mutated to F, (6) V at position 389 mutated to I, (7) A at position 485 mutated to E, (8) K at position 584 mutated to E, (9) V at position 589 mutated to H, (10) L at position 408 mutated to I, and (11) Y at position 409 mutated to A.

[0021] According to some examples of the present application, the inventors found that a DNA polymerase having one of the above mutation types (or any combination thereof) has higher enzyme activity.

[0022] According to an embodiment of the application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has: an amino acid mutation at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589, or a functionally equivalent site; and an amino acid mutation at at least one of the following 15 sites and functionally equivalent sites: position 382, ​​397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725.

[0023] In some examples of the present application, compared to the wild-type KOD DNA polymerase, the DNA polymerase mutant has: an amino acid mutation at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589, or a functionally equivalent position; and an amino acid mutation at at least one of the following 14 positions and functionally equivalent positions: position 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725.

[0024] In some examples of the present application, the inventors found that polymerase mutants having amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589, or functionally equivalent positions (SEQ ID NO: 1) have higher enzyme activity. The inventors found that mutations at one or more positions selected from positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725 based on the amino acid sequence shown in SEQ ID NO: 1 can obtain mutants with different enzyme activities.

[0025] According to an embodiment of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has: an amino acid mutation at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or a functionally equivalent site; and an amino acid mutation at at least one site selected from the following four sites and functionally equivalent sites: position 397, position 424, position 481, and position 553.

[0026] In some examples of the present application, based on the amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions, mutations at one or more positions selected from positions 397, 424, 481 and 553 can obtain mutants with higher enzyme activity.

[0027] According to the embodiments of the present application, the mutation types of the mutation sites are: (1) D at position 141 mutated to A, (2) E at position 143 mutated to A, (3) H at position 147 mutated to E, (4) S at position 383 mutated to T, (5) Y at position 384 mutated to F, (6) V at position 389 mutated to I, (7) A at position 485 mutated to E, (8) K at position 584 mutated to E, (9) V at position 589 mutated to H, (10) L at position 408 mutated to I, (11) Y at position 409 mutated to A, (12) W at position 397 mutated to Y or H, (13) N at position 424 mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S, (14) D at position 432 mutated to R, E, H or M, (15) F mutated to R, (16) M at position 523 mutated to R or L, (17) A at position 553 mutated to T, R, N, E, G, H, L, M, F, Y, P, S, D, V, I or C, (18) M at position 561 mutated to R or W, (19) L at position 564 mutated to R or M, (20) Q at position 461 mutated to I, L, M, F, W or Y, (21) Y at position 1 mutates to R, H, L, M or F, (22) T at position 605 mutates to I or V, (23) H at position 663 mutates to R, Q, L, M, F, T, Y, N, V, G, A, P, S, C, E or K, (24) G at position 711 mutates to R, M or S, (25) H at position 725 mutates to R, (26) Q at position 382 mutates to R, H, L or Y.

[0028] In some examples of the present application, the mutation types of the mutation sites are: (1) D at position 141 mutates to A, (2) E at position 143 mutates to A, (3) H at position 147 mutates to E, (4) S at position 383 mutates to T, (5) Y at position 384 mutates to F, (6) V at position 389 mutates to I, (7) A at position 485 mutates to E, (8) K at position 584 mutates to E, (9) V at position 589 mutates to H, (10) L at position 408 mutates to I, (11) Y at position 409 mutates to A, (12) W at position 397 mutates to Y or H, (13) 3) N at position 424 mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S (14) D at position 432 mutated to R, E, H or M, (15) F at position 445 mutated to R, (16) M at position 523 mutated to R or L, (17) A at position 553 mutated to T, R, N, E, G, H, L, M, F, Y, P, S, D, V, I or C, (18) M at position 561 mutated to R or W, (19) L at position 564 mutated to R or M, (20) Q at position 461 mutated to I, L, M, F, W or Y, (21) The Y at position mutated to R, H, L, M or F, (22) the T at position 605 mutated to I or V, (23) the H at position 663 mutated to R, Q, L, M, F, T, Y, N, V, G, A, P, S, C, E or K, (24) the G at position 711 mutated to R, M or S, (25) the H at position 725 mutated to R.

[0029] In some examples of the present application, the inventors found that DNA polymerase mutants with the above mutation types still have different degrees of enzymatic activity.

[0030] According to an embodiment of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at one position selected from the following 15 positions and functionally equivalent positions: positions 382, ​​397, 424, 436, 440, 451, 463, 471, 483, 490, 508, 512, 524, 536, 540, 551, 561, 570, 581, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 610, 611, 612, 623, 634, 645, 650, 661, 673, 682, 684, 691, 702, 713, 724, 735, 740, 751, 762, 774, 785, 791, 803, 814, 825 The invention also provides that the Q at position 382 cannot be mutated to R, H, L and Y; or the N at position 424 cannot be mutated to R and L; or the D at position 432 cannot be mutated to M; or the H at position 663 cannot be mutated to T, V, P and E.

[0031] It should be noted that the so-called Q at position 382 cannot mutate to R, H, L, or Y means that the Q at position 382 cannot mutate to R, or the Q at position 382 cannot mutate to H, or the Q at position 382 cannot mutate to L, or the Q at position 382 cannot mutate to Y. Similarly, the N at position 424 cannot mutate to R and L means that the N at position 424 cannot mutate to R, or the N at position 424 cannot mutate to L. In this application, this expression method is also applicable to any embodiment. It will not be repeated in the following embodiments or implementation methods.

[0032] According to the embodiment of the present application, the wild type KOD Compared with the DNA polymerase, the DNA polymerase mutant has: an amino acid mutation at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or a functionally equivalent site; and an amino acid mutation at one of the following 14 sites and functionally equivalent sites: position 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; provided that the N at position 424 cannot be mutated to R and L; or the D at position 432 cannot be mutated to M; or the H at position 663 cannot be mutated to T, V, P and E.

[0033] According to the embodiment of the present application, the wild type KOD Compared with the DNA polymerase described above, the DNA polymerase mutant has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at two positions selected from the following 14 positions and functionally equivalent positions: 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725; provided that, when H at position 663 mutates to R, A at position 553 cannot mutate to P or E; or when H at position 663 mutates to Q, Q at position 461 cannot mutate to Y.

[0034] In some examples of the present application, when the H at position 663 of the mutant is mutated to R, and the A at position 553 is mutated to P or E; or, when the H at position 663 is mutated to Q, and the Q at position 461 is mutated to Y, the enzyme activity is low.

[0035] According to an embodiment of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has mutations (1)-(11), and any one mutation selected from (12)-(25).

[0036] In some examples of the present application, the amino acid sequence of the DNA polymerase mutant having mutations (1)-(11) is shown in SEQ ID NO:1.

[0037] In some examples of the present application, a polymerase mutant having the amino acid sequence shown in SEQ ID NO: 1 was used as a control group for polymerization activity detection.

[0038] In some examples of the present application, a DNA polymerase mutant having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 has higher enzymatic activity. In some preferred examples, it has at least 91% identity, or at least 92% identity, or at least 93% identity, or at least 94% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity, and in some most preferred examples, it has at least 100% identity.

[0039] According to an embodiment of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has mutations (1)-(11); and any one mutation selected from the following mutations: W at position 397 mutates to H, N at position 424 mutates to Q, A at position 553 mutates to R, A at position 553 mutates to N, A at position 553 mutates to E, A at position 553 mutates to G, A at position 553 mutates to H, A at position 553 mutates to P, A at position 553 mutates to S, Q at position 461 mutates to W, Q at position 461 mutates to Y, Y at position 481 mutates to M, Y at position 481 mutates to F, G at position 711 mutates to R, and G at position 711 mutates to S.

[0040] In some examples of the present application, the inventors have experimentally verified that further amino acid single-point mutations based on the amino acid sequence shown in SEQ ID NO: 1 can further increase the enzyme polymerization activity.

[0041] According to an embodiment of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has mutations (1)-(11); and any one mutation combination selected from the following mutation combinations:

[0042] The 481st Y mutated to F, and the 553rd A mutated to P; the 481st Y mutated to F, and the 553rd A mutated to S; the 397th W mutated to H, and the 553rd A mutated to P; the 461st Q mutated to Y, and the 553rd A mutated to P; the 481st Y mutated to F, and the 553rd A mutated to G; the 424th N mutated to Q, and the 553rd A mutated to P; the 397th W mutated to H, and the 481st Y mutated to F; the 553rd A mutated to P, and the 711th G mutated to R; the 397th W mutated to H, and the 553rd A mutated to S; the 481st Y mutated to M, and the 553rd A mutated to P; the 461st Q mutated to W, and the 553rd A mutated to P; A at position 553 mutated to P, and G at position 711 mutated to S; Q at position 461 mutated to Y, and A at position 553 mutated to S; A at position 553 mutated to P, and M at position 561 mutated to W; A at position 553 mutated to P, and M at position 561 mutated to R; Y at position 481 mutated to F, and G at position 711 mutated to R; Q at position 461 mutated to F, and A at position 553 mutated to P; N at position 424 mutated to Q, and A at position 553 mutated to S; D at position 432 mutated to H, and A at position 553 mutated to P; A at position 553 mutated to P, and T at position 605 mutated to V; Y at position 481 mutated to F, and M at position 561 mutated to W; N at position 424 mutated to F, and the 553rd A mutated to P; the 481st Y mutated to F, and the 605th T mutated to I; the 461st Q mutated to F, and the 481st Y mutated to F; the 553rd A mutated to P, and the 663rd H mutated to R; the 481st Y mutated to M, and the 553rd A mutated to S; the 553rd A mutated to P, and the 564th L mutated to M; the 432nd D mutated to E, and the 553rd A mutated to P; the 461st Q mutated to F, and the 553rd A mutated to S; the 481st Y mutated to M, and the 553rd A mutated to G; the 481st Y mutated to F, and the 564th L mutated to M; the 553rd A mutated to P, and the 663rd H mutated to N; D mutated to H, and A at position 553 mutated to S; A at position 553 mutated to P, and H at position 663 mutated to F; A at position 553 mutated to P, and L at position 564 mutated to R; N at position 424 mutated to S, and A at position 553 mutated to P; Y at position 481 mutated to F, and A at position 553 mutated to F; A at position 553 mutated to E, and H at position 663 mutated to R; Y at position 481 mutated to L, and G at position 711 mutated to R; N at position 424 mutated to Q, and H at position 725 mutated to R; N at position 424 mutated to Q, and H at position 663 mutated to R; Q at position 461 mutated to W, and M at position 561 mutated to R; D at position 432 mutated to R, and A at position 553 mutated to S;W at position 397 mutated to H, and H at position 663 mutated to Y; Q at position 461 mutated to Y, and H at position 663 mutated to Q; Q at position 461 mutated to I, and Y at position 481 mutated to M; A at position 553 mutated to Y, and T at position 605 mutated to V; N at position 424 mutated to K, and H at position 663 mutated to L; Q at position 461 mutated to F, and Y at position 481 mutated to R.;

[0043] According to the examples of this application, some mutants may have base bias in sequencing applications and can be adaptively selected based on actual experimental needs.

[0044] According to an embodiment of the present application, compared with the wild-type KOD DNA polymerase, the DNA polymerase mutant has mutations (1)-(11), and any one mutation combination selected from the following mutation combinations: Y at position 481 mutates to F, and A at position 553 mutates to P; Y at position 481 mutates to F, and A at position 553 mutates to S; W at position 397 mutates to H, and A at position 553 mutates to P; Q at position 461 mutates to Y, and A at position 553 mutates to P; N at position 424 mutates to Q, and A at position 553 mutates to P; W at position 397 mutates to H, and Y at position 481 mutates to F; A at position 553 mutates to P, and G at position 711 mutates to R; W at position 397 mutates to H, and A at position 553 mutates to P; A mutated to S; Y mutated to F, and G mutated to R; Q mutated to F, and Y mutated to F; A mutated to P, and L mutated to M; Y mutated to F, and L mutated to M; N mutated to S, and A mutated to P; Y mutated to L, and G mutated to R; N mutated to Q, and H mutated to R; W mutated to H, and H mutated to Y; Q mutated to I, and Y mutated to M.

[0045] In some examples of the present application, the inventors have experimentally verified that double-site amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 1 can further increase the enzyme polymerization activity.

[0046] In the second aspect of the present application, the present application provides a nucleic acid molecule encoding the DNA polymerase mutant described in the first aspect of the present application. The DNA polymerase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.

[0047] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0048] In the third aspect of the present application, the present application provides an expression vector, wherein the expression vector comprises or carries the nucleic acid molecule described in the second aspect of the present application. In the present application, the type of expression vector is not particularly limited, as long as it can replicate and express the corresponding mutant in the host cell.

[0049] It should be noted that the expression vector may further include a promoter, and the promoter is operably connected to the nucleic acid molecule.

[0050] According to an embodiment of the present application, the expression vector is a non-pathogenic viral vector, and the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.

[0051] According to an embodiment of the present application, the expression vector is a non-viral vector, and the non-viral vector includes but is not limited to a plasmid vector.

[0052] In a fourth aspect of the present application, the present application provides a recombinant cell, the recombinant cell carrying the nucleic acid molecule described in the second aspect of the present application and the expression vector described in the third aspect of the present application. The recombinant cell is used to express or secrete the DNA polymerase mutant described in the first aspect of the present application.

[0053] According to an embodiment of the present application, the recombinant cell is selected from Escherichia coli, yeast and mammalian cells.

[0054] In some examples of the present application, the recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently express the above-mentioned DNA polymerase mutant under appropriate conditions.

[0055] In a fifth aspect of the present application, a recombinant strain is provided, wherein the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the present application. The DNA polymerase mutant can be obtained quickly and in large quantities by culturing the recombinant strain.

[0056] In the sixth aspect of the present application, the present application proposes a method for obtaining a DNA polymerase mutant, which comprises culturing the recombinant cell described in the fourth aspect of the present application or the recombinant strain described in the fifth aspect under conditions suitable for protein expression to obtain the DNA polymerase mutant.

[0057] In the seventh aspect of the present application, the present application proposes a complex, which includes the DNA polymerase mutant described in the first aspect of the present application and a small molecule compound or a macromolecule, and the DNA polymerase mutant and the small molecule compound or the macromolecule are coupled through a chemical bond.

[0058] According to an embodiment of the present application, the small molecule compound or macromolecule includes a fluorescent marker, fluorescein or an antibody, etc.

[0059] It should be noted that the application scenarios of the DNA polymerase mutants and complexes proposed in this application include but are not limited to nucleic acid synthesis and nucleic acid sequencing. They can also be used to screen drugs targeting viruses or cell division, and have great development prospects in forensic medicine and criminology.

[0060] In an eighth aspect of the present application, a method for nucleic acid synthesis is provided, comprising: subjecting a mixture of a nucleic acid template, an amplification primer, dNTPs, and the DNA polymerase mutant described in the first aspect of the present application to an amplification treatment under conditions suitable for nucleic acid amplification, thereby obtaining the nucleic acid. The aforementioned method for nucleic acid synthesis enables efficient and rapid amplification of the nucleic acid template.

[0061] It should be noted that the DNA polymerase mutant described in the present application has polymerization activity for all dNTPs (including dNTPs with or without fluorescent labels).

[0062] In the ninth aspect of the present application, the present application proposes a method for nucleic acid sequencing, which comprises: subjecting a nucleic acid template to be tested and a mixed product of the DNA polymerase mutant and modified dNTPs described in the first aspect of the present application to an amplification treatment and a fluorescence signal detection treatment under conditions suitable for nucleic acid amplification; and determining the nucleic acid sequence of the nucleic acid to be tested based on the fluorescent signal obtained by detection.

[0063] Exemplarily, the nucleic acid sequencing method includes mixing the nucleic acid template to be tested with the DNA polymerase mutant and the non-natural dNTP with a fluorescently labeled 3'O-reversible terminator, wherein the DNA polymerase mutant is responsible for matching the non-natural dNTP with the fluorescently labeled 3'O-reversible terminator with the nucleic acid template, and finally, by detecting multiple fluorescent labeling signals, the nucleic acid sequence obtained based on the obtained fluorescent labeling signals is obtained to obtain the nucleic acid sequence of the nucleic acid to be tested. The above polymerization reaction and fluorescent labeling reaction can be performed for multiple cycles according to the length of the sequencing template.

[0064] In the tenth aspect of the present application, the present application provides a nucleic acid sequencing kit, which comprises the DNA polymerase mutant described in the first aspect or the complex described in the seventh aspect. The kit described in the present application is used for efficient, accurate and rapid nucleic acid sequencing.

[0065] In the eleventh aspect of the present application, the present application proposes a use of the nucleic acid sequencing kit of the tenth aspect in sequencing. According to an embodiment of the present application, the kit can be used for sequencing, including but not limited to sequencing by synthesis (SBS).

[0066] In the twelfth aspect of the present application, the present application proposes a use of the DNA polymerase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the recombinant strain described in the fifth aspect, or the complex described in the seventh aspect in the preparation of a product for catalyzing DNA amplification or nucleic acid sequencing. According to embodiments of the present application, the DNA polymerase mutant, nucleic acid molecule, expression vector, recombinant cell, recombinant strain, or complex can be used alone or in combination to prepare a product for catalyzing DNA amplification or nucleic acid sequencing.

[0067] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0069] FIG1 is a schematic diagram of a recombinant KOD DNA polymerase expression plasmid pET22b-KOD plasmid according to an embodiment of the present application;

[0070] FIG2 is a schematic diagram of the expression and bacterial lysate results of wild-type and some mutant KOD DNA polymerases described in an example of the present application;

[0071] FIG3 is a schematic diagram showing the results of detecting the activities of wild-type and some mutant KOD DNA polymerases using the FRET method described in one embodiment of the present application. DETAILED DESCRIPTION

[0072] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0073] In this application, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specified.

[0074] In this application, unless otherwise indicated, the term "amino acid" is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0075] In this application, unless otherwise indicated, the term "identity" has the conventional meaning in the art and refers to the "homology" between two nucleic acid or amino acid sequences, wherein the percentage represents the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences to be compared after optimal alignment, with the differences between the two sequences being randomly distributed over their entire lengths. In this application, the mutants are described according to their mutations at specific residues, the positions of which are located with reference to the positions of the amino acids in the amino acid sequence of wild-type KOD polymerase set forth in SEQ ID NO: 2.

[0076] The KOD DNA polymerase mutant (SEQ ID NO: 1) is obtained by mutating the wild-type KOD DNA polymerase (SEQ ID NO: 2). With reference to the sequence position of SEQ ID NO: 2, the mutation sites include: D at position 141 is mutated to A, E at position 143 is mutated to A, H at position 147 is mutated to E, S at position 383 is mutated to T, Y at position 384 is mutated to F, V at position 389 is mutated to I, L at position 408 is mutated to I, Y at position 409 is mutated to A, A at position 485 is mutated to E, K at position 584 is mutated to E, and V at position 589 is mutated to H.

[0077] In this application, unless otherwise specified, the term "relative polymerization activity" refers to the polymerization activity of each experimental group relative to the reference group, calculated when the polymerization activity of the reference group is assumed to be 100%. For example, in the Examples of this application, the reference group DNA polymerase referred to herein is DP01, whose amino acid sequence is shown in SEQ ID NO: 1.

[0078] In this application, unless otherwise indicated, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of genes caused by the introduction of exogenous DNA into cells.

[0079] In this application, unless otherwise specified, amino acid sequences are displayed from 5' end to 3' end.

[0080] In this application, unless otherwise specified, non-natural dNTPs or modified dNTPs have the same meaning, including but not limited to dNTPs with labels (eg, fluorescent labels) and / or dNTPs with an O-reversible terminator at the 3' end.

[0081] In this application, unless otherwise stated, the polymerase mutants used in the examples of this application are all polymerase mutant fusion proteins with 6 His tags connected to the C-terminus. As known to those skilled in the art, they can all be replaced by untagged polymerase mutants.

[0082] Studies have reported that thermostable B-family polymerases from thermophilic archaea perform well in incorporating natural nucleotides or their analogs in high-throughput sequencing. These thermostable B-family polymerases include KOD (Thermococcus kodakaraensis), 9°N (Thermococcus sp. 9°N), TGO (Thermococcus gorgonarius), TOK (Desulfurococcus sp. Tok), Vent DNA polymerase (Thermococcus litoralis), JDF-3, and pfu DNA polymerase (Pyrococcus furiosis).

[0083] To improve the polymerization speed of existing DNA polymerases and increase reaction read lengths in high-throughput sequencing processes (such as sequencing by synthesis (SBS)), the inventors have discovered through in-depth research that the incorporation efficiency of modified dNTPs can be enhanced by enzyme engineering of KOD DNA polymerase.

[0084] The inventors protected the functional domains of the wild-type polymerase to ensure it could still perform its original basic functions. By conducting kinetic simulations and statistical inference on the palm, finger, and thumb regions of the KOD DNA polymerase, they identified mutation sites suitable for experimental screening. Furthermore, through extensive experimental verification, they ultimately obtained a DNA polymerase suitable for attachment to DNBs (DNA nanoballs) on chip surfaces.

[0085] The present invention will be described below with reference to examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0086] Example 1: Preparation of KOD DNA polymerase mutants

[0087] In this example, the amino acid sequence of KOD DNA polymerase (DP01) is shown in SEQ ID NO: 1.

[0088] 1. Construction of DP01 expression vector

[0089] The recombinant expression vector pET22b-WT was constructed by recombining the His-tag fused KOD DNA polymerase encoding gene into the pET22b vector according to the instructions of the Seamless Cloning Kit (Manufacturer: Norwegian; Catalog No.: C112-01). The resulting vector contained the His-tag fused KOD DNA polymerase encoding gene (Figure 1), and expression was induced by IPTG.

[0090] The nucleotide sequence of the KOD DNA polymerase encoding gene fused with a His tag is the sequence obtained by connecting six His tag codons to the 3' end of SEQ ID NO: 3;

[0091] The amino acid sequence of the KOD DNA polymerase fusion protein is obtained by connecting six His tags to the C-terminus of the amino acids shown in SEQ ID NO: 1.

[0092] 2. Construction of recombinant bacteria

[0093] The recombinant expression vector pET22b-WT was introduced into competent E. coli BL21 cells (Beijing Solaibao Technology Co., Ltd.). Positive colonies were screened on resistance plates (containing 50 μg / ml ampicillin). Three to five positive monoclonal colonies were selected and identified by PCR using primers SQF (Table 1) and SQR (Table 1). A positive clone was identified by obtaining a fragment of 2500 bp, which was essentially consistent with the theoretical value. This positive clone was designated BL21 / pET22b-WT.

[0094] Table 1: Primer sequence information

[0095] 3. Expression of DP01 and preparation of crude extract

[0096] Pick a single BL21 / pET22b-WT colony and culture it in 150 μl LB liquid medium (containing 50 μg / ml ampicillin) at 37°C, 200 rpm, overnight. The next day, dilute it 1:100 and transfer it to 150 μl LB liquid medium (containing 50 μg / ml ampicillin) at 37°C, 220 rpm, and shake culture until the OD 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 37°C overnight. The induced BL21 / pET22b-WT bacterial culture was collected. At the same time, no IPTG was added as a blank control, and the uninduced BL21 / pET22b-WT bacterial culture was collected.

[0097] After induction, add 1 μM PMSF (protease inhibitor) and 50 μg lysozyme to the BL21 / pET22b-WT bacterial suspension, mix well, and incubate at 37°C for 10 minutes to lyse the bacteria. This is the crude fusion protein extract. This crude protein extract can be used for subsequent protein activity assays.

[0098] The crude extract of the lysed KOD DNA polymerase fusion protein was subjected to SDS-PAGE (5% stacking gel, 12% separating gel). The results are shown in FIG2 . Lane 1 is a protein marker (Page Ruler Prestained Protein Ladder, 26616, Thermo Fisher Scientific), lane 12 is 5 μl of KOD DNA polymerase fusion protein (DP01) + 5 μl of 2X loading buffer, and lanes 2-11 are 5 μl of KOD DNA polymerase mutant fusion proteins (DP001, DP002, DP004, DP005, DP007, DP008, DP009, DP024, DP011, DP012) + 5 μl of 2X loading buffer. It can be seen that the protein in lanes 2-12 is approximately 91.5 kDa in size, which is consistent with the molecular weight reported in the literature (Huber C, Marx A. Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension). selectivity.PLoS One.2017 Aug 23;12(8):e0183623; Chim N, Shi C, Sau SP, Nikoomanzar A, Chaput JC. Structural basis for TNA synthesis by an engineered TNA polymerase. Nat Commun.2017Nov 27;8(1):1810.).

[0099] The uninduced BL21 / pET22b-WT bacterial suspension did not yield the approximately 91.5 kDa target protein. The empty pET22b vector was introduced into E. coli BL21 to generate BL21 / pET22b. Expression and lysis using the above method also failed to yield the approximately 91.5 kDa target protein.

[0100] Example 2: Preparation of KOD DNA polymerase mutant fusion protein

[0101] In this embodiment, the KOD DNA polymerase mutant fusion protein is a protein obtained by subjecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all fourteen of amino acids 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725 of the amino acid sequence of DP01 (SEQ ID NO: 1) to amino acid mutations. If only one amino acid is mutated, the resulting protein is a single-point mutant of the KOD DNA polymerase; if two amino acids are mutated, the resulting protein is a two-point combination mutant of the KOD DNA polymerase, and so on.

[0102] The KOD DNA polymerase mutant encoding gene is a nucleic acid obtained by subjecting the nucleotide sequence of the KOD DNA polymerase mutant DP01 encoding gene (SEQ ID NO: 3) to amino acid codon mutations at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all fourteen amino acid codons among positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725 in its corresponding amino acid sequence (SEQ ID NO: 1).

[0103] In the mutant KOD DNA polymerase protein, the amino acid positions are mutated as follows:

[0104] The W at position 397 mutated to Y or H;

[0105] The N at position 424 is mutated to R, Q, H, I, L, M, F, W, K, Y, V, C, or S;

[0106] The D at position 432 mutated to R, E, H, or M;

[0107] F at position 445 mutated to R;

[0108] The M at position 523 mutated to R or L;

[0109] The A at position 553 is mutated to T, R, N, E, G, H, L, M, F, Y, P, S, D, V, I, or C;

[0110] The M at position 561 mutated to R or W;

[0111] L at position 564 mutated to R or M;

[0112] Q at position 461 mutated to I, L, M, F, W, or Y;

[0113] Y at position 481 mutated to R, H, L, M, or F;

[0114] T at position 605 mutated to I or V;

[0115] H mutation at position 663 to R, Q, L, M, F, T, Y, N, V, G, A, P, S, C, E, or K;

[0116] G at position 711 mutated to R, M, or S;

[0117] The H at position 725 was mutated to R.

[0118] 1. Single point mutation KOD DNA polymerase mutant protein

[0119] The amino acid sequence of KOD DNA polymerase SEQ ID NO: 1 was mutated at one of the 14 amino acids 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725, while the other amino acid sequences remained unchanged, to obtain a protein having DNA polymerase activity (Table 2).

[0120] Table 2: Mutation and mutation information of KOD DNA polymerase single point mutants

[0121] 2. Double-point mutation KOD DNA polymerase mutant protein

[0122] Two amino acids selected from 14 positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725 in the amino acid sequence of the KOD DNA polymerase SEQ ID NO: 1 are mutated, while the other amino acid sequences remain unchanged, to obtain a protein having DNA polymerase activity:

[0123] The amino acid sequence of DP80 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to P;

[0124] The amino acid sequence of DP81 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to S;

[0125] The amino acid sequence of DP82 is as follows: W at position 397 of SEQ ID NO: 1 is mutated to H, and A at position 553 is mutated to P;

[0126] The amino acid sequence of DP83 is as follows: Q at position 461 of SEQ ID NO: 1 is mutated to Y, and A at position 553 is mutated to P;

[0127] The amino acid sequence of DP84 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to G;

[0128] The amino acid sequence of DP85 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to Q, and A at position 553 is mutated to P;

[0129] The amino acid sequence of DP86 is as follows: W at position 397 of SEQ ID NO: 1 is mutated to H, and Y at position 481 is mutated to F;

[0130] The amino acid sequence of DP87 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and G at position 711 is mutated to R;

[0131] The amino acid sequence of DP88 is as follows: W at position 397 of SEQ ID NO: 1 is mutated to H, and A at position 553 is mutated to S;

[0132] The amino acid sequence of DP89 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to M, and A at position 553 is mutated to P;

[0133] The amino acid sequence of DP90 is as follows: the Q at position 461 of SEQ ID NO: 1 is mutated to W, and the A at position 553 is mutated to P;

[0134] The amino acid sequence of DP91 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and G at position 711 is mutated to S;

[0135] The amino acid sequence of DP92 is as follows: Q at position 461 of SEQ ID NO: 1 is mutated to Y, and A at position 553 is mutated to S;

[0136] The amino acid sequence of DP93 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and M at position 561 is mutated to W;

[0137] The amino acid sequence of DP94 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and M at position 561 is mutated to R;

[0138] The amino acid sequence of DP96 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and G at position 711 is mutated to R;

[0139] The amino acid sequence of DP97 is as follows: Q at position 461 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to P;

[0140] The amino acid sequence of DP98 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to Q, and A at position 553 is mutated to S;

[0141] The amino acid sequence of DP99 is as follows: the 432nd D of SEQ ID NO: 1 is mutated to H, and the 553rd A is mutated to P;

[0142] The amino acid sequence of DP100 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and T at position 605 is mutated to V;

[0143] The amino acid sequence of DP101 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and M at position 561 is mutated to W;

[0144] The amino acid sequence of DP102 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to P;

[0145] The amino acid sequence of DP103 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and T at position 605 is mutated to I;

[0146] The amino acid sequence of DP104 is as follows: Q at position 461 of SEQ ID NO: 1 is mutated to F, and Y at position 481 is mutated to F;

[0147] The amino acid sequence of DP105 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and H at position 663 is mutated to R;

[0148] The amino acid sequence of DP106 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to M, and A at position 553 is mutated to S;

[0149] The amino acid sequence of DP107 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and L at position 564 is mutated to M;

[0150] The amino acid sequence of DP108 is as follows: the 432nd position D of SEQ ID NO: 1 is mutated to E, and the 553rd position A is mutated to P;

[0151] The amino acid sequence of DP109 is as follows: Q at position 461 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to S;

[0152] The amino acid sequence of DP110 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to M, and A at position 553 is mutated to G;

[0153] The amino acid sequence of DP111 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and L at position 564 is mutated to M;

[0154] The amino acid sequence of DP112 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and H at position 663 is mutated to N;

[0155] The amino acid sequence of DP113 is as follows: the 432nd D of SEQ ID NO: 1 is mutated to H, and the 553rd A is mutated to S;

[0156] The amino acid sequence of DP114 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and H at position 663 is mutated to F;

[0157] The amino acid sequence of DP115 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to P, and L at position 564 is mutated to R;

[0158] The amino acid sequence of DP116 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to S, and A at position 553 is mutated to P;

[0159] The amino acid sequence of DP117 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to F, and A at position 553 is mutated to F;

[0160] The amino acid sequence of DP118 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to E, and H at position 663 is mutated to R;

[0161] The amino acid sequence of DP119 is as follows: Y at position 481 of SEQ ID NO: 1 is mutated to L, and G at position 711 is mutated to R;

[0162] The amino acid sequence of DP120 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to Q, and H at position 725 is mutated to R;

[0163] The amino acid sequence of DP121 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to Q, and H at position 663 is mutated to R;

[0164] The amino acid sequence of DP122 is as follows: the 461st Q of SEQ ID NO: 1 is mutated to W, and the 561st M is mutated to R;

[0165] The amino acid sequence of DP123 is as follows: the 432nd position D of SEQ ID NO: 1 is mutated to R, and the 553rd position A is mutated to S;

[0166] The amino acid sequence of DP124 is as follows: W at position 397 of SEQ ID NO: 1 is mutated to H, and H at position 663 is mutated to Y;

[0167] The amino acid sequence of DP126 is as follows: the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the H at position 663 is mutated to Q;

[0168] The amino acid sequence of DP127 is as follows: the 461st Q of SEQ ID NO: 1 is mutated to I, and the 481st Y is mutated to M;

[0169] The amino acid sequence of DP128 is as follows: A at position 553 of SEQ ID NO: 1 is mutated to Y, and T at position 605 is mutated to V;

[0170] The amino acid sequence of DP130 is as follows: N at position 424 of SEQ ID NO: 1 is mutated to K, and H at position 663 is mutated to L;

[0171] The amino acid sequence of DP131 is as follows: the 461st Q in SEQ ID NO: 1 is mutated to F, and the 481st Y is mutated to R.

[0172] 3. Preparation of recombinant vector expressing KOD DNA polymerase mutant

[0173] Recombinant vectors expressing different KOD DNA polymerase point mutants were prepared by using the KOD DNA polymerase vector pET22b-WT as a template and performing at least one site-directed mutagenesis to obtain each mutant using the instructions of the Mut Express II Fast Mutagenesis Kit V2 (manufacturer: Novazonics; Catalog No.: C214-02). The protein-encoding genes of the different KOD DNA polymerase point mutants fused with a His tag were recombined into the pET22b vector. The resulting vectors, incorporating the protein-encoding genes of the different point mutants fused with a His tag, were expressed by inducing IPTG.

[0174] The amino acid sequence of each KOD DNA polymerase point mutant fusion protein is based on SEQ ID NO: 1 with point mutations and six His tags attached to its C-terminus. As known in the art, different enzymes can be tagged at either the N-terminus or the C-terminus to facilitate purification. Tags typically follow the sequences shown in Table 3. The presence or absence of tags has no effect on enzyme performance.

[0175] Table 3: Tag sequences

[0176] 4. Construction of recombinant bacteria

[0177] The construction steps were the same as those in Example 1. The recombinant vectors expressing different KOD DNA polymerase point mutants prepared in step 3 were introduced into BL21 to obtain recombinant bacteria expressing different KOD DNA polymerase mutant fusion proteins.

[0178] 5. Mutant Expression and Crude Extract Preparation

[0179] The preparation steps are the same as those in Example 1. The recombinant bacteria expressing different KOD DNA polymerase point mutant fusion proteins prepared in step 4 are expressed and lysed to obtain crude extracts of different KOD DNA polymerase point mutant fusion proteins.

[0180] SDS-PAGE gel electrophoresis was used to detect crude extracts of different KOD DNA polymerase point mutant fusion proteins. The results are shown in FIG2 , indicating that proteins with good homogeneity were obtained.

[0181] Example 3: Performance testing of recombinant KOD DNA polymerase mutant fusion protein

[0182] The point mutant fusion proteins in this example were expressed and cleaved according to the method in Example 1.

[0183] 1. Single-base incorporation kinetics of KOD DNA polymerase point mutant fusion proteins

[0184] In this example, dATP (dATP-AF532) and dTTP (dTTP-AF532) labeled with AF532 fluorescent dye, and a DNA template (template DNA-AF647) labeled with AF647 fluorescent dye were used to simulate the incorporation of modified nucleotides during SBS sequencing. The relative reaction rates of recombinant KOD DNA polymerase mutants were measured using a microplate reader, and the activity ratio of each mutant relative to KOD polymerase DP01 was measured. The specific experimental method is as follows:

[0185] Single-stranded primers S1A (Table 1) and S2A (Table 1) with 5' AF647 fluorescent label (synthesized by Sangon Biotech Co., Ltd.) were mixed at an equimolar concentration of 1:1, annealed at 80°C for 10 min, and the annealed product was stored at -20°C in the dark to obtain template DNA-AF647 labeled with AF647 fluorescent dye.

[0186] Enzyme activity was detected using a TECAN microplate reader. The reaction was carried out in 96-well plates (Corning black, clear bottom 96 plates) with a total reaction volume of 50 μl.

[0187] The reaction system is: 20 μl KOD polymerase mutant fusion protein crude extract, 0.25 μM dATP-AF532, 0.25 μM dTTP, 0.25 μM dCTP, 0.25 μM dGTP, 0.1 μM template DNA-AF647 for the experiment, the enzyme reaction buffer is 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton, pH 8.8; the reaction temperature is 40°C.

[0188] The enzyme reaction was carried out in kinetic detection mode, and data were recorded every minute. The detection conditions were as shown in Table 4.

[0189] Table 4: Enzyme kinetics assay reaction conditions

[0190] After the reaction is completed, the data table or enzyme activity curve can be directly exported, and the reaction rate of its relative fluorescence value can be approximately calculated.

[0191] Polymerization reactions were performed using KOD DNA polymerase fusion protein (DP01) and KOD DNA polymerase point mutant fusion protein as examples, wherein the C-terminus of the fusion protein contained six his tags.

[0192] For detailed information on the KOD DNA polymerase point mutants, refer to Table 2. For preparation methods, refer to Examples 1 and 2.

[0193] The DP01 fusion protein is a highly active KOD DNA polymerase, with a relative polymerization activity that is 2-3 times higher than that of a polymerase containing only mutations 408 and 409 (see Patent No. PCT / CN2019 / 102493). Based on the highly active variants, this application further optimizes and improves the polymerization activity of KOD DNA polymerase mutants. The activity assay kinetic curves of some KOD DNA polymerase mutants using single-stranded DNA as a template and dATP-AF532 as a substrate are shown in Figure 3.

[0194] The polymerization activity test results of the wild-type KOD DNA polymerase, the polymerase with only mutations 408 and 409, and each KOD DNA polymerase mutant relative to the polymerization activity of KOD DNA polymerase DP01 are shown in Table 5. Compared with KOD DNA polymerase (DP01), mutants DP003, DP011, DP012, DP013, DP014, DP019, DP020, DP023, DP024, DP041, DP83, DP112, DP113, DP115, DP117, and DP130 showed comparable polymerization activity; mutants DP001, DP002, DP004, DP005, DP006, DP007, DP008, DP009, DP010, DP015, DP016, and DP017 showed comparable polymerization activity. 7. DP018, DP021, DP022, DP80, DP81, DP82, DP83, DP84, DP85, DP86, DP87, DP88, DP89, DP91, DP92, DP93, DP96, DP101, DP104, DP106, DP107, DP108, DP109, DP110, DP111, DP116, DP119, DP120, DP121, DP122, DP123, DP124, DP127, etc. showed better polymerization activity.

[0195] Table 5: Kinetic test results of KOD DNA polymerase mutants

[0196] Note: Since WT (wild-type KOD DNA polymerase) cannot incorporate modified dATP, the relative polymerization activity is 0.

[0197] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0198] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A DNA polymerase mutant, characterized in that, compared with the wild-type KOD DNA polymerase, the mutant has amino acid mutations at position 408, position 409 or functionally equivalent positions, and amino acid mutations at at least 1 position selected from the following 23 positions and functionally equivalent positions: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589, position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; the amino acid sequence outside the amino acid mutation sites of the mutant has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase; the wild-type KOD DNA polymerase has the amino acid sequence shown in SEQ ID NO:

2.

2. The DNA polymerase mutant according to claim 1, characterized in that, the amino acid sequence outside the amino acid mutation sites of the mutant is the same as the corresponding amino acid sequence of the wild-type KOD DNA polymerase.

3. The DNA polymerase mutant according to claim 1, characterized in that, compared with the wild-type KOD DNA polymerase, the mutant has amino acid mutations at position 408, position 409 or functionally equivalent positions, and amino acid mutations at at least 1 position selected from the following 9 positions and functionally equivalent positions: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589.

4. The DNA polymerase mutant according to claim 3, characterized in that, compared with the wild-type KOD DNA polymerase, the mutant has amino acid mutations at position 408, position 409 or functionally equivalent positions, and amino acid mutations at at least 1 position selected from the following 2 positions and functionally equivalent positions: position 147, position 584.

5. The DNA polymerase mutant according to claim 3 or 4, characterized in that, the mutation types at the mutation sites are: (1) D at position 141 is mutated to A, (2) E at position 143 is mutated to A, (3) H at position 147 is mutated to E, (4) S at position 383 is mutated to T, (5) Y at position 384 is mutated to F, (6) V at position 389 is mutated to I, (7) A at position 485 is mutated to E, (8) K at position 584 is mutated to E, (9) V at position 589 is mutated to H, (10) L at position 408 is mutated to I, (11) Y at position 409 is mutated to A.

6. The DNA polymerase mutant according to claim 1, characterized in that, compared with the wild-type KOD DNA polymerase, the mutant has: Amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions, and Amino acid mutations at at least one position selected from the following 14 positions and functionally equivalent positions: Position 397, 424, 432, 445, 523, 553, 561, 564, 461, Position 481, 605, 663, 711, 725.

7. The DNA polymerase mutant according to claim 6, wherein, compared with the wild-type KOD DNA polymerase, the mutant has: Amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and Amino acid mutations at at least one position selected from the following 4 positions and functionally equivalent positions: Position 397, 424, 481, 553.

8. The DNA polymerase mutant according to claim 6, wherein, the mutation types at the mutation sites are: (1) D at position 141 is mutated to A, (2) E at position 143 is mutated to A, (3) H at position 147 is mutated to E, (4) S at position 383 is mutated to T, (5) Y at position 384 is mutated to F, (6) V at position 389 is mutated to I, (7) A at position 485 is mutated to E, (8) K at position 584 is mutated to E, (9) V at position 589 is mutated to H, (10) L at position 408 is mutated to I, (11) Y at position 409 is mutated to A, (12) W at position 397 is mutated to Y or H, (13) N at position 424 is mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S, (14) D at position 432 is mutated to R, E, H or M, (15) F at position 445 is mutated to R, (16) M at position 523 is mutated to R or L, (17) A at position 553 is mutated to T, R, N, E, G, H, L, M, F, Y, P, S, D, V, I or C, (18) M at position 561 is mutated to R or W, (19) L at position 564 is mutated to R or M, (20) Q at position 461 is mutated to I, L, M, F, W or Y, (21) Y at position 481 is mutated to R, H, L, M or F, (22) T at position 605 is mutated to I or V, (23) H at position 663 is mutated to R, Q, L, M, F, T, Y, N, V, G, A, P, S, C, E or K, (24) G at position 711 is mutated to R, M or S, (25) H at position 725 is mutated to R.

9. The DNA polymerase mutant according to claim 6, wherein, compared with the wild-type KOD DNA polymerase, the mutant has: Amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and An amino acid mutation at one position selected from the following 14 positions and functionally equivalent positions: Position 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; Provided that the N at position 424 cannot mutate to R and L; or The D at position 432 cannot mutate to M; or The H at position 663 cannot mutate to T, V, P and E.

10. The DNA polymerase mutant according to claim 6, characterized in that compared with the wild-type KOD DNA polymerase, the mutant has: Amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and Amino acid mutations at two positions selected from the following 14 positions and functionally equivalent positions: Position 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; Provided that when the H at position 663 mutates to R, the A at position 553 cannot mutate to P and E; or When the H at position 663 mutates to Q, the Q at position 461 cannot mutate to Y.

11. The DNA polymerase mutant according to claim 8, characterized in that compared with the wild-type KOD DNA polymerase, the mutant has the mutations of (1)-(11) and any one of the mutations selected from (12)-(25).

12. The DNA polymerase mutant according to claim 11, characterized in that compared with the wild-type KOD DNA polymerase, the mutant has the mutations of (1)-(11) and any one of the following mutations: The W at position 397 mutates to H, The N at position 424 mutates to Q, The A at position 553 mutates to R, The A at position 553 mutates to N, The A at position 553 mutates to E, The A at position 553 mutates to G, The A at position 553 mutates to H, The A at position 553 mutates to P, The A at position 553 mutates to S, The Q at position 461 mutates to W, The Q at position 461 mutates to Y, The Y at position 481 mutates to M, The Y at position 481 mutates to F, The G at position 711 mutates to R, The G at position 711 mutates to S.

13. The DNA polymerase mutant according to claim 8, characterized in that Compared with the wild-type KOD DNA polymerase, the mutant has the mutations of (1)-(11), and any one of the following mutant combinations: Y at position 481 is mutated to F, and A at position 553 is mutated to P; Y at position 481 is mutated to F, and A at position 553 is mutated to S; W at position 397 is mutated to H, and A at position 553 is mutated to P; Q at position 461 is mutated to Y, and A at position 553 is mutated to P; Y at position 481 is mutated to F, and A at position 553 is mutated to G; N at position 424 is mutated to Q, and A at position 553 is mutated to P; W at position 397 is mutated to H, and Y at position 481 is mutated to F; A at position 553 is mutated to P, and G at position 711 is mutated to R; W at position 397 is mutated to H, and A at position 553 is mutated to S; Y at position 481 is mutated to M, and A at position 553 is mutated to P; Q at position 461 is mutated to W, and A at position 553 is mutated to P; A at position 553 is mutated to P, and G at position 711 is mutated to S; Q at position 461 is mutated to Y, and A at position 553 is mutated to S; A at position 553 is mutated to P, and M at position 561 is mutated to W; A at position 553 is mutated to P, and M at position 561 is mutated to R; Y at position 481 is mutated to F, and G at position 711 is mutated to R; Q at position 461 is mutated to F, and A at position 553 is mutated to P; N at position 424 is mutated to Q, and A at position 553 is mutated to S; D at position 432 is mutated to H, and A at position 553 is mutated to P; A at position 553 is mutated to P, and T at position 605 is mutated to V; Y at position 481 is mutated to F, and M at position 561 is mutated to W; N at position 424 is mutated to F, and A at position 553 is mutated to P; Y at position 481 is mutated to F, and T at position 605 is mutated to I; Q at position 461 is mutated to F, and Y at position 481 is mutated to F; A at position 553 is mutated to P, and H at position 663 is mutated to R; Y at position 481 is mutated to M, and A at position 553 is mutated to S; A at position 553 is mutated to P, and L at position 564 is mutated to M; D at position 432 is mutated to E, and A at position 553 is mutated to P; Q at position 461 is mutated to F, and A at position 553 is mutated to S; Y at position 481 is mutated to M, and A at position 553 is mutated to G; Y at position 481 is mutated to F, and L at position 564 is mutated to M; A at position 553 is mutated to P, and H at position 663 is mutated to N; D at position 432 is mutated to H, and A at position 553 is mutated to S; A at position 553 is mutated to P, and H at position 663 is mutated to F; A at position 553 is mutated to P, and L at position 564 is mutated to R; N at position 424 is mutated to S, and A at position 553 is mutated to P; Y at position 481 is mutated to F, and A at position 553 is mutated to F; A at position 553 is mutated to E, and H at position 663 is mutated to R; Y at position 481 is mutated to L, and G at position 711 is mutated to R; N at position 424 is mutated to Q, and H at position 725 is mutated to R; The 424th N is mutated to Q, and the 663rd H is mutated to R; The 461st Q is mutated to W, and the 561st M is mutated to R; The 432nd D is mutated to R, and the 553rd A is mutated to S; The 397th W is mutated to H, and the 663rd H is mutated to Y; The 461st Q is mutated to Y, and the 663rd H is mutated to Q; The 461st Q is mutated to I, and the 481st Y is mutated to M; The 553rd A is mutated to Y, and the 605th T is mutated to V; The 424th N is mutated to K, and the 663rd H is mutated to L; The 461st Q is mutated to F, and the 481st Y is mutated to R.

14. The DNA polymerase mutant according to claim 13, wherein, compared with the wild-type KOD DNA polymerase, the mutant has the mutations of (1)-(11) and any one of the following mutation combinations: The 481st Y is mutated to F, and the 553rd A is mutated to P; The 481st Y is mutated to F, and the 553rd A is mutated to S; The 397th W is mutated to H, and the 553rd A is mutated to P; The 461st Q is mutated to Y, and the 553rd A is mutated to P; The 424th N is mutated to Q, and the 553rd A is mutated to P; The 397th W is mutated to H, and the 481st Y is mutated to F; The 553rd A is mutated to P, and the 711th G is mutated to R; The 397th W is mutated to H, and the 553rd A is mutated to S; The 481st Y is mutated to F, and the 711th G is mutated to R; The 461st Q is mutated to F, and the 481st Y is mutated to F; The 553rd A is mutated to P, and the 564th L is mutated to M; The 481st Y is mutated to F, and the 564th L is mutated to M; The 424th N is mutated to S, and the 553rd A is mutated to P; The 481st Y is mutated to L, and the 711th G is mutated to R; The 424th N is mutated to Q, and the 725th H is mutated to R; The 397th W is mutated to H, and the 663rd H is mutated to Y; The 461st Q is mutated to I, and the 481st Y is mutated to M.

15. A nucleic acid molecule, wherein, the nucleic acid molecule encodes the DNA polymerase mutant according to any one of claims 1-14.

16. An expression vector, wherein, the expression vector contains or carries the nucleic acid molecule according to claim 15.

17. The expression vector according to claim 16, wherein, the expression vector is a non-pathogenic viral vector or a non-viral vector, and the non-pathogenic viral vector includes an adenovirus vector or a retrovirus vector; the non-viral vector includes a plasmid vector.

18. A recombinant cell, wherein, the recombinant cell carries the nucleic acid molecule according to claim 15 or the expression vector according to any one of claims 16-17.

19. The recombinant cell according to claim 18, wherein, the recombinant cell is selected from Escherichia coli, yeast and mammalian cells.

20. A recombinant strain, wherein, The recombinant strain expresses the DNA polymerase mutant according to any one of claims 1 to 14.

21. A method for obtaining a DNA polymerase mutant, characterized in that the method comprises: culturing and treating the recombinant cell according to any one of claims 18 to 19 or the recombinant strain according to claim 20 under conditions suitable for protein expression so as to obtain the DNA polymerase mutant.

22. A complex, characterized in that the complex comprises the DNA polymerase mutant according to any one of claims 1 to 14 and a small molecule compound or a macromolecule, and the mutant and the small molecule compound or macromolecule are coupled by a chemical bond.

23. The complex according to claim 22, characterized in that the small molecule compound or macromolecule comprises a fluorescent label, fluorescein or an antibody.

24. A nucleic acid synthesis method, characterized in that the method comprises: performing an amplification treatment on a mixture of a nucleic acid template, an amplification primer, dNTP and the DNA polymerase mutant according to any one of claims 1 to 14 under conditions suitable for nucleic acid amplification so as to obtain the nucleic acid.

25. A nucleic acid sequencing method, characterized in that the method comprises: performing an amplification treatment and a fluorescence signal detection treatment on a mixture of a nucleic acid to be measured, the DNA polymerase mutant according to any one of claims 1 to 14 and a modified dNTP under conditions suitable for nucleic acid amplification; and determining the nucleic acid sequence of the nucleic acid to be measured based on the detected fluorescence signal.

26. A nucleic acid sequencing kit, characterized in that the kit comprises the DNA polymerase mutant according to any one of claims 1 to 14 or the complex according to any one of claims 22 to 23.

27. Use of the nucleic acid sequencing kit according to claim 26 in sequencing.

28. Use of the DNA polymerase mutant according to any one of claims 1 to 14, the nucleic acid molecule according to claim 15, the expression vector according to any one of claims 16 to 17, the recombinant cell according to any one of claims 18 to 19, the recombinant strain according to claim 20 or the complex according to any one of claims 22 to 23 in the preparation of products related to catalyzing DNA amplification or nucleic acid sequencing.

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