DNA polymerases and uses thereof

Abstract

This application discloses a DNA polymerase and its applications. The DNA polymerase of this application has an amino acid sequence that is at least 80% identical to that of the 9°N DNA polymerase, and the DNA polymerase includes at least one type I amino acid substitution mutation at positions A675, G677, E734, and P739 or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme. When used for nucleic acid sequencing, the DNA polymerase of this application can significantly reduce the error rate, improve the accuracy, increase the Q value, reduce phase errors, and improve the overall quality of nucleic acid sequencing. The DNA polymerase of this application provides a new scheme and approach for high-quality nucleic acid sequencing, which is of great significance to nucleic acid sequencing.

Description

Technical Field

[0001] This application relates to the field of nucleic acid sequencing technology, and in particular to a DNA polymerase and its applications. Background Technology

[0002] With the rapid development of sequencing technology, the requirements for active molecules in sequencing systems have also increased. Currently, the mainstream Sequencing by Synthesis (SBS) method follows the base complementary pairing principle, extending one base with fluorescent and termination structures in each round under enzyme catalysis. After capturing the base information of each target strand, the dye and termination structure are removed for the next round of reaction, and this process is repeated to obtain sequence information. Because sequencing uses non-natural modified bases, the sequencing enzyme (such as DNA polymerase) does not perfectly match the pocket region where it binds, thus affecting sequencing efficiency. Sequencing primers also retain chemical groups on their non-natural bases during sequencing, which can significantly reduce sequencing efficiency and speed.

[0003] Therefore, obtaining thermostable DNA polymerases with stronger binding ability and higher fidelity to sequencing bases remains a key research focus in the field of nucleic acid sequencing technology. Summary of the Invention

[0004] The purpose of this application is to provide a new DNA polymerase and its application, which aims to solve at least one of the above-mentioned technical problems to some extent.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] The first aspect of this application discloses a DNA polymerase having an amino acid sequence that is at least 80% identical to the amino acid sequence of a 9°N DNA polymerase (UniProtID: Q56366; PDB ID: 5OMQ), and the DNA polymerase includes at least one Class I amino acid substitution mutation at positions A675, G677, E734, and P739 or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme.

[0007] The second aspect of this application discloses a nucleic acid molecule that encodes the DNA polymerase of this application.

[0008] A third aspect of this application discloses an expression vector containing the nucleic acid molecule of this application.

[0009] The fourth aspect of this application discloses a host cell containing the expression vector of this application.

[0010] In this application, the host cell refers to a cell or microorganism capable of expressing the expression vector of this application and obtaining the DNA polymerase of this application. For example, in one implementation of this application, the expression vector is transfected into bacteria, that is, bacteria are used as the host of the expression vector of this application.

[0011] The fifth aspect of this application discloses the application of the DNA polymerase of this application in nucleic acid sequencing.

[0012] It should be noted that the DNA polymerase of this application, as a DNA polymerase, can be applied to routine in vitro nucleic acid amplification, such as PCR; it can also further meet the needs of nucleic acid sequencing, such as for generating complementary strands of nucleic acid templates.

[0013] The sixth aspect of this application discloses a sequencing kit containing the DNA polymerase of this application.

[0014] It should be noted that the sequencing kit of this application contains the DNA polymerase of this application, and may also contain other components for sequencing. Other components, such as dNTPs, buffers, reversible terminators, etc., can be found in existing nucleic acid sequencing reactions. Other components of the kit can be purchased separately, but for ease of use, they can also be combined into the kit of this application.

[0015] It should also be noted that one of the purposes of this application is to improve the DNA polymerase so that it can better meet the needs of nucleic acid sequencing. Its essence is to be able to stably and efficiently perform nucleic acid extension in vitro. Therefore, the DNA polymerase of this application can also be used to prepare nucleic acid extension kits, such as PCR kits.

[0016] In one implementation of this application, the reagent kit includes nucleotides.

[0017] In one implementation of this application, the nucleotide includes a reversible terminator.

[0018] In one implementation of this application, the reversible terminator comprises a purine or pyrimidine base and a ribose or deoxyribose moiety, the ribose or deoxyribose moiety having a removable 3'-OH blocking group covalently linked thereto.

[0019] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0020] The DNA polymerase described in this application can significantly reduce the error rate, improve the accuracy, and increase the Q value when used for nucleic acid sequencing. It can also reduce phase errors (phasing and pre-phasing) in sequencing, thereby improving the overall quality of nucleic acid sequencing. This provides a new approach and method for high-quality nucleic acid sequencing and is of great significance to nucleic acid sequencing. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the experimental process for immobilizing nucleic acid complexes on the surface of magnetic beads in this application embodiment;

[0022] Figure 2 These are the Slow-Reaction and Fast-Reaction test results for some mutants in the embodiments of this application;

[0023] Figure 3 These are the Slow-Reaction and Fast-Reaction test results for another subset of mutants in the embodiments of this application;

[0024] Figure 4 These are some of the mutant sequencing data analysis results from the embodiments of this application;

[0025] Figure 5 This is another part of the mutant sequencing data analysis results in the embodiments of this application;

[0026] Figure 6 This is a schematic diagram of sites G677, A675, E734, P739 and their side chain structures in an embodiment of this application;

[0027] Figure 7 This is a schematic diagram of the mutant side chain structure of G677R, A675R, E734R, and P739R in the embodiments of this application. Detailed Implementation

[0028] The present application will now be described in further detail with reference to specific embodiments and accompanying drawings. The following experiments are merely illustrative and should not be construed as limiting the present application. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other devices, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; a complete understanding of the related operations can be obtained based on the description in the specification and general technical knowledge in the art.

[0029] In this application, the term "sequencing" is also referred to as "nucleic acid sequencing" or "gene sequencing," and these three terms are interchangeable in meaning, all referring to the determination of the type and sequence of bases or nucleotides (including nucleotide analogs) in nucleic acid molecules. The sequencing process includes binding nucleotides to a template and collecting the corresponding signals emitted by the nucleotides (including analogs). The sequencing process includes sequencing by synthesis (sequencing-while-synthesizing, SBS) and / or sequencing by ligation (sequencing-while-ligating, SBL), including DNA sequencing and / or RNA sequencing, including long-fragment sequencing and / or short-fragment sequencing. The terms "long-fragment" and "short-fragment" are relative; for example, nucleic acid molecules longer than 1Kb, 2Kb, 5Kb, or 10Kb can be called long fragments, and those shorter than 1Kb or 800bp are called short fragments.

[0030] Sequencing generally involves multiple rounds to determine the type and sequence of multiple bases or nucleotides on a nucleic acid template. This application refers to each round of sequencing as a "cycle." A "cycle" of sequencing, also called a "sequencing round," is defined as one base extension of four nucleotides / bases, or as determining the type of a base or nucleotide at any specified position on the template. For sequencing platforms based on polymerization or ligation reactions, a cycle of sequencing includes the process of binding four nucleotides (including nucleotide analogs) to the nucleic acid template via base complementarity and collecting the corresponding signals. Specifically, for platforms based on polymerization reactions, the reaction system includes reaction substrate nucleotides, polymerase, and a nucleic acid template. The nucleic acid template has a sequence (sequencing primer) attached. Based on base pairing principles and polymerization reaction principles, the added reaction substrate nucleotides, catalyzed by the polymerase, are ligated to the sequencing primers, achieving the binding of the nucleotide to a specific position on the nucleic acid template. Typically, a sequencing run may include one or more base extensions (repeat). For example, four nucleotides are added to the reaction system sequentially, and base extensions and corresponding reaction signal acquisition are performed separately, resulting in four base extensions in one sequencing run. Alternatively, four nucleotides may be added to the reaction system in any combination, such as in pairs, with each pair performing base extensions and corresponding reaction signal acquisition separately, resulting in two base extensions in one sequencing run. Yet another example is that four nucleotides are added to the reaction system simultaneously for base extensions and reaction signal acquisition, resulting in one base extension in one sequencing run.

[0031] In this application, the term "nucleic acid molecule" means: a polymeric form of nucleotides of any length, and may include ribonucleotides or analogs thereof, deoxyribonucleotides or analogs thereof, and mixtures of the above nucleotides or analogs thereof. The term "nucleic acid molecule" may refer to a single-stranded polynucleotide or a double-stranded polynucleotide. Nucleotides in a nucleic acid molecule may include naturally occurring nucleotides and their functionally substituted analogs. Examples of analogs can hybridize with nucleic acids in a sequence-specific manner or can be used as templates for the replication of specific nucleotide sequences. Naturally occurring nucleotides typically have a backbone comprising phosphodiester bonds. Analog structures may have alternative backbone linkages including any kind known in the art. Naturally occurring nucleotides typically have deoxyribose (e.g., found in DNA) or ribose (e.g., found in RNA). Analog structures may have alternative sugar moieties, including any kind known in the art. Nucleotides may include natural or non-natural bases. The bases in natural DNA may include one or more of adenine, thymine, cytosine, and / or guanine, and the bases in natural RNA may include one or more of adenine, uracil, cytosine, and / or guanine. Nucleotides may also use any non-natural bases or base analogues, such as locked nucleic acids (LNAs) and bridging nucleic acids (BNAs).

[0032] In this application, the term "nucleic acid template" refers to a parent nucleic acid molecule or fragment thereof that binds to nucleotides or nucleotide analogs through successive rounds of base extension. The nucleic acid template can be the entire sequence of a nucleic acid molecule or a partial fragment thereof. The "nucleic acid template" can be a nucleic acid fragment used as a template in a sequencing-while-synthesizing reaction. Since the bases added in the extension reaction satisfy the base pairing principle with the sequencing template, the sequence of the sequencing template can be determined by identifying the type of bases added in each round of extension. The sequence of the nucleic acid template here typically includes, but is not limited to, at least one of the target molecule sequence, UMI sequence, and sample tag sequence; it is sometimes also referred to as an "insertion fragment" or "target molecule" in this application.

[0033] In this application, the term "primer" refers to an oligonucleotide or nucleic acid molecule capable of hybridizing with a target sequence of interest. In embodiments, the primer acts as a substrate to which nucleotides can be polymerized by a polymerase. For example, a primer can serve as a starting point for DNA or RNA synthesis. For instance, sequencing primers can hybridize with a nucleic acid template strand to initiate the synthesis of a new strand complementary to the template strand. Primers can comprise any combination of nucleotides or the like. In some instances, primers are single-stranded oligonucleotides or polynucleotides.

[0034] In this application, the term "reversible terminator" refers to a nucleotide or analogue that blocks the formation of a covalent bond between the 3' hydroxyl portion of a nucleotide or analogue's carbon sugar and the 5' phosphate of another nucleotide or analogue. The blocking portion on the nucleotide or analogue can be reversible, thus allowing the 3' hydroxyl group to form a covalent bond with the 5' phosphate of another nucleotide by removing or modifying the blocking portion. For example, a "reversible terminator" may include a blocking portion located, for example, at the 3' position of a nucleotide, and may be a chemically cleavable portion, such as allyl, azidomethyl, or methoxymethyl, or may be an enzyme-cleavable group, such as a phosphate ester. A "reversible terminator" may or may not have a detectable label.

[0035] In this application, the term "reversible terminator sequencing" refers to the process of sequencing using reversible terminators as dNTPs.

[0036] In this application, the term "phase decoupling," also known as "phase imbalance," "phase decoupling," or "phase difference," refers to the phenomenon of asynchronous reactions among nucleic acid molecules in a group, such as a nucleic acid molecule cluster, during a chemical reaction. This includes phase lag (phasing or sequence lag) and phase lead (prephasing or sequence lead). Phase lag (phasing or sequence lag) refers to a nucleotide analog that should react and be incorporated into the nucleic acid template in cycle N, but instead participates in the reaction in cycle N+1. Phase lead (prephasing or sequence lead) refers to a nucleotide analog that should react and be incorporated into the nucleic acid template in cycle N, but instead participates in the reaction in cycle N-1, i.e., crosstalk occurs between adjacent cycles within the same channel. This phase error accumulates and intensifies with increasing sequencing cycle number. The final result is that all four nucleotides are present simultaneously and with uniform brightness within an amplification cluster. In this situation, the base recognition algorithm will be unable to identify the correct sequencing signal in that round, meaning it will be unable to accurately obtain the sequence information of the nucleotide molecule to be tested.

[0037] In this application, the "+" sign between amino acid sequence sites means "and", indicating that the sites before and after the "+" sign coexist.

[0038] This application provides a DNA polymerase having an amino acid sequence that is at least 80% identical to the amino acid sequence of a 9°N DNA polymerase, and the DNA polymerase includes at least one of the first-class amino acid substitution mutations at positions A675, G677, E734, and P739 or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme.

[0039] In one implementation of this application, the first type of amino acid substitution mutation includes at least one of the substitution mutations of A675R, A675K, G677R, G677K, E734K, E734R, P739K, and P739R or functionally equivalent substitution mutations.

[0040] In one implementation of this application, the first type of amino acid substitution mutation includes one of the following mutations:

[0041] (a)A675R / K;

[0042] (b)G677R / K;

[0043] (c)E734R / K;

[0044] (d)P739R / K;

[0045] (e) Combination mutations of L631M and E734R / K;

[0046] (f) Combinatorial mutations of G677R / K, A675R / K and E734R / K;

[0047] (g)A675R / K and E734R / K combined mutations;

[0048] (h) Combination mutations of G677R / K and E734R / K;

[0049] (i) Combined mutations of E734R / K and P739R / K.

[0050] In one implementation of this application, the DNA polymerase further includes a second type of amino acid substitution mutation at at least one of the following positions or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme: D141, E143, C223, L408, Y409, P410, L478, A485, Y497, and H633.

[0051] In one implementation of this application, the second type of amino acid substitution mutation includes at least one of the substitution mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, and H633G or functionally equivalent substitution mutations.

[0052] In one implementation of this application, the second type of amino acid substitution mutation includes one of the following mutations:

[0053] (a) Combination mutations of D141A, C223S and Y497G;

[0054] (b) Combination mutations of D141A, L408A, Y409A, P410I and H633G;

[0055] (c) Combination mutations of D141A, L408A, Y409A, P410I and H633G;

[0056] (d) Combination mutations of L408A, Y409A, P410I, L478S, A485L and H633G;

[0057] (e) Combination mutations of D141A, C223S, L408A, Y409A, L478S, A485L and H633G;

[0058] (f) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G and H633G.

[0059] In one implementation of this application, the DNA polymerase further includes at least one third-class amino acid substitution mutation at the T622, L631 position or a functionally equivalent position on the 9°N amino acid sequence of the sequencing enzyme.

[0060] In one implementation of this application, the third type of amino acid substitution mutation includes at least one of the T622A, L631M substitution mutations or functionally equivalent substitution mutations.

[0061] In one implementation of this application, the third type of amino acid substitution mutation includes a combination mutation of T622A and L631M.

[0062] In one implementation of this application, the DNA polymerase further includes at least one type IV amino acid substitution mutation at one of the following positions or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme: M129, M329, P410, E576, T590, T590, H633, K705, I521, V278, K559, E599, L408, R247, T349, E580, S407, and V471.

[0063] In one implementation of this application, the fourth type of amino acid substitution mutation includes at least one of the following substitution mutations or functionally equivalent substitution mutations: M129A, M329H, P410L, E576L, T590K, T590R, H633T, K705A, I521L, V278L, K559G, E599D, L408S, R247Y, T349R, E580K, S407A, and V471G.

[0064] In one implementation of this application, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme:

[0065] (a) Combination mutations of L631M, E734R and I521L;

[0066] (b) Combination mutations of D141A, L408A, Y409A, P410I, H633G, L631M, E734R and I521L;

[0067] (c) Combination mutations of D141A, C223S, L631M, E734R and I521L;

[0068] (d) Combination mutations of D141A, L408A, Y409A, P410I, C223S, L631M, E734R and I521L;

[0069] (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, L631M, E734R and I521L;

[0070] (f) Combination mutations of V278L, L631M, E734R and I521L;

[0071] (g) Combination mutations of L408A, Y409A, P410I, V278L, L631M, E734R and I521L;

[0072] (h) Combination mutations of L408A, Y409A, P410I, L478S, A485L, V278L, L631M, E734R and I521L;

[0073] (i) Combination mutations of D141A, L408A, Y409A, P410I, L478S, A485L, H633G, V278L, L631M, E734R and I521L;

[0074] (j) Combinatorial mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, L631M, E734R and I521L;

[0075] (k)L631M, E734R, I521L and E599D combined mutations;

[0076] (l) Combination mutations of L408A, Y409A, P410I, L631M, E734R, I521L and E599D;

[0077] (m) Combination mutations of E143A, C223S, L408A, Y409A, A485L, Y497G, L631M, E734R, I521L and E599D;

[0078] (n) Combination mutations of D141A, E143A, C223S, L408A, Y409A, A485L, Y497G, L631M, E734R, I521L and E599D;

[0079] (o) Combination mutations of E143A, C223S, L408A, Y409A, A485L, Y497G, L631M, E734R, I521L and E599D;

[0080] (p) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, L631M, E734R, I521L and E599D;

[0081] (q) Combination mutations of D141A, C223S, L408A, Y409A, A485L, L631M, E734R, I521L and E599D;

[0082] (r) Combination mutations of L408A, Y409A, P410I, L631M, E734R, I521L and E599D;

[0083] Combination mutations of (s)D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, L631M, E734R, I521L and E599D.

[0084] In one implementation of this application, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme:

[0085] (a) Combined mutations of K559G and E580K;

[0086] (b) Combination mutations of L408A, Y409A, P410I, L478S, A485L, Y497G, K559G and E580K;

[0087] (c) Combination mutations of D141A, L408A, Y409A, P410I, A485L, K559G and E580K;

[0088] (d) Combinatorial mutations of L408A, Y409A, P410I, K559G and E580K;

[0089] (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, K559G and E580K.

[0090] In one implementation of this application, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme:

[0091] (a) Combined mutations of E734R and I521L;

[0092] (b) Combination mutations of E143A, C223S, L408A, Y409A, P410I, A485L, Y497G, E734R and I521L;

[0093] (c) Combination mutations of D141A, L408A, Y409A, P410I, A485L, E734R and I521L;

[0094] (d) Combination mutations of L408A, Y409A, P410I, E734R and I521L;

[0095] (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, E734R and I521L.

[0096] In one implementation of this application, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme:

[0097] (a) Combination mutations of E734R, I521L and E599D;

[0098] (b) Combination mutations of L408A, Y409A, P410I, A485L, Y497G, H633G, E734R, I521L and E599D;

[0099] (c) Combination mutations of D141A, E143A, C223S, L408A, Y409A, A485L, E734R, I521L and E599D;

[0100] (d) Combination mutations of L408A, Y409A, P410I, E734R, I521L and E599D;

[0101] (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, E734R, I521L and E599D.

[0102] In one implementation of this application, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme:

[0103] (a) Combination mutations of A675R, S407A and V471G;

[0104] (b) Combination mutations of C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, A675R, S407A and V471G;

[0105] (c) Combination mutations of E143A, C223S, L408A, Y409A, P410I, L478S, A675R, S407A and V471G;

[0106] (d) Combination mutations of L408A, Y409A, P410I, L478S, A675R, S407A and V471G;

[0107] (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, A675R, S407A and V471G;

[0108] (f) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G and E734R.

[0109] In one implementation of this application, the DNA polymerase is a group B DNA polymerase.

[0110] In one implementation of this application, the polymerase is selected from group B archaea DNA polymerase, human DNA polymerase-a, T4 polymerase, RB69 polymerase, or phi29 phage DNA polymerase.

[0111] In one implementation of this application, the group B archaea DNA polymerase is selected from the genera *Thermococcus*, *Vorococcus*, or *Methanococcus*.

[0112] The DNA polymerase provided in this application can be obtained in any way, without strict limitations. As one implementation method, it can be obtained through screening using the mutant screening method developed by the inventors for DNA polymerase.

[0113] To more efficiently screen large numbers of monoclonal sequencing enzyme mutants with unpredictable functions, this application develops a method for screening sequencing enzyme mutants, including a pre-screening culture step, a first detection step, a screening culture step, and a second detection step.

[0114] The pre-screening culture step includes: placing multiple mutant monoclonal antibodies of the target sequencing enzyme in a bacterial culture medium for fermentation, adding a protein expression inducer during fermentation to induce mutant expression, and then lysing the bacterial culture to obtain bacterial lysate.

[0115] In this application embodiment, there is no limitation on the number of mutant single clones. The method provided in this application embodiment is particularly suitable for experiments on batch samples with a large number of mutants and active functional sites. The source of the multiple mutant single clones can be mutant single clones obtained by random mutation, mutant single clones obtained by a combination of mutation at a specified site and random mutation, or a mixture of both.

[0116] In this embodiment, multiple mutant single clones of the target sequencing enzyme are placed in bacterial culture medium for fermentation. This method enables batch culture of multiple mutant single clones and allows for batch operation during the induction step, achieving high-throughput screening.

[0117] In one implementation of this application, the fermentation process involves placing multiple mutant monoclonal samples of the target sequencing enzyme in a bacterial culture medium. The fermentation includes two stages: primary seed fermentation and secondary seed fermentation. Primary seed fermentation allows the mutant to rapidly multiply within a short time, yielding a large number of clones. Secondary seed fermentation increases fermentation density and seed vigor, ultimately improving the production efficiency of the mutant and ensuring its fermentation effect.

[0118] During secondary seed fermentation, a protein expression inducer can be added to induce mutant expression. In some embodiments, the protein expression inducer is selected from at least one of isopropyl-β-D-thiogalactoside and L-arabinose.

[0119] For example, the pre-screening culture step specifically includes the following operations:

[0120] 1. Transformation: The mutant clone was transformed, and BL21 DE3 competent cells were cultured after being transformed with 100 ng of plasmid.

[0121] 2. Primary Seed Fermentation: On the second day, select single clones and add them to a 96-well plate to culture the mutant bacterial culture, with 200 μL of bacterial culture per well. Culture the primary seed overnight at 37°C and 200 rpm. This step requires sealing the 96-well plate with sealing film to prevent liquid evaporation.

[0122] 3. Secondary seed fermentation and induction of target protein expression: The fermented primary seed was added to LB medium in 96-well plates at an inoculation ratio of 1:100 (200 μL per well) and cultured at 37°C and 200 rpm for 3 h. Subsequently, 500 μM IPTG (isopropyl-β-D-thiogalactoside) was added, and the target protein expression was induced overnight at 25°C and 200 rpm.

[0123] 4. Bacterial lysis and protein efflux: Add lysis buffer to the bacterial culture in a 1:10 ratio in a 96-well plate, and incubate at room temperature with shaking at 20 rpm for 1 hour to lyse the target protein and obtain the bacterial lysis buffer.

[0124] The first detection step includes: using bacterial lysis buffer to simulate reversible terminator sequencing, and screening for preliminary mutants based on the detection results.

[0125] It is understandable that this step directly uses bacterial lysate to simulate reversible terminator sequencing, which cannot control the actual amount of the target protein reacting. Therefore, it is only used as a means to screen out mutants with low activity and obtain preliminary screening mutants.

[0126] In one implementation of this application, the reversible termination reaction of sequencing includes: (1) providing a nucleic acid complex immobilized on the surface of a solid support, the nucleic acid complex comprising a nucleic acid template and sequencing primers whose sequences at least partially bind to the nucleic acid template; (2) adding a solution comprising bacterial lysis buffer and a reversible terminator, and contacting the solution with the nucleic acid complex under polymerization reaction conditions; (3) detecting the reaction efficiency of each analyte in the polymerization reaction, and evaluating the activity of the analyte based on the reaction efficiency.

[0127] This application's embodiments simulate a reversible termination sequencing reaction in solution, which can completely replicate the real sequencing environment of the sequencing enzyme, in order to screen for sequencing enzyme mutants with better activity in real sequencing.

[0128] In step (1), the solid-phase support is understood as a carrier or support for immobilizing nucleic acid molecules or nucleic acid templates. In some application examples, the solid-phase support may also be referred to as a solid substrate, sequencing chip, or sequencing biochip. For example, the solid-phase support may be a substrate, magnetic beads, microspheres, or nanoparticles.

[0129] In this embodiment, a nucleic acid template is immobilized on the surface of a solid-phase support, and the nucleic acid template is attached to the surface of the solid-phase support. In some embodiments, a probe is attached to the surface of the solid-phase support, and the probe is covalently linked to a molecular / group on the surface of the solid-phase support, with the nucleic acid template attached to the surface of the solid-phase support via the probe. The probe is an oligonucleotide or nucleic acid molecule fragment capable of hybridizing with a target nucleic acid molecule of interest. In one embodiment, at least a portion of the probe is configured to hybridize with at least a portion of the 3' end of the nucleic acid template. In another embodiment, one end of the nucleic acid template is covalently linked to a molecular / group on the surface of the solid-phase support, thereby attaching to the surface of the solid-phase support. The nucleic acid template can further bind sequencing primers via base complementarity; in one embodiment, the probe acts as a sequencing primer, forming a nucleic acid complex with the nucleic acid template.

[0130] In step (2), the bacterial lysis buffer is derived from the lysis buffer obtained by lysing the bacterial cells in the pre-screening culture step, and it replaces the sequencing enzyme required for the sequencing reaction. The reversible terminators include reversible terminators dATP, dTTP, dGTP, and dCTP. Furthermore, the reaction solution used to simulate the pseudo-terminator sequencing also contains synthetic reagents, including but not limited to buffer salts and magnesium ions. In some embodiments, the solution also includes monovalent metal ions such as sodium or potassium ions and ammonium ions. It should be understood that in the embodiments of this application, the bacterial lysis buffer derived from each mutant clone is prepared with the pseudo-terminator solution and then contacted with the nucleic acid complex.

[0131] In this embodiment, the solution is contacted with the nucleic acid complex under suitable conditions for polymerization. Under these conditions, the nucleic acid complex, the lysing protein in the bacterial lysate, and the reversible terminator come into contact. However, when the bacterial lysate contains a polymerase-active protein that can promote the polymerization of the pseudoterminator with the nucleic acid complex, the pseudoterminator can bind to the nucleic acid complex and polymerize at the 3' end of the sequencing primer chain to extend the length by one nucleotide.

[0132] After the predetermined reaction time, the unreacted reversible terminator is removed by washing.

[0133] In step (3), the reaction efficiency of each analyte in the polymerization reaction is detected, and the activity of each bacterial lysate is evaluated based on the obtained reaction efficiency.

[0134] The reaction efficiency of polymerization of each analyte in this application embodiment can be detected by various methods. As an example, at least one of the following methods can be used:

[0135] 1) The sequencing primers in the nucleic acid complex of step (1) are made to carry fluorescent groups. In some examples, fluorescent groups are introduced at the 3' end of the sequencing primers. A fluorescence quenching group is introduced into the reversible terminator in step (2). In this step, under the action of bacterial lysis buffer, if the reversible terminator binds to the 3' end of the sequencing primer, the fluorescence quenching group carried by the reversible terminator will cause fluorescence quenching of the sequencing primer; if the reversible terminator does not bind to the 3' end of the sequencing primer, the signal generated by the fluorescent group carried by the sequencing primer can be detected before and after the polymerization reaction. In step (3), the change in fluorescence intensity before and after the polymerization reaction is detected, and the reaction efficiency of the polymerization reaction is analyzed based on the change in fluorescence intensity. Under the same polymerization reaction conditions, the greater the decrease in fluorescence signal intensity after the polymerization reaction relative to the fluorescence signal intensity before the polymerization reaction, the higher the reaction efficiency of the polymerization reaction.

[0136] 2) The sequencing primers in step (1) are made to carry a fluorescence quenching group. In some examples, a fluorescence quenching group is introduced at the 3' end of the sequencing primers. A fluorescent group is introduced into the reversible terminator in step (2). In this step, under the action of the bacterial lysis buffer, if the reversible terminator binds to the 3' end of the sequencing primer, the fluorescence quenching group carried by the sequencing primer will cause the fluorescence of the reversible terminator to be quenched; if the reversible terminator does not bind to the 3' end of the sequencing primer, the signal generated by the fluorescent group carried by the reversible terminator bound to the nucleic acid complex can be detected. In step (3), the fluorescence intensity after the polymerization reaction is detected, and the reaction efficiency of the polymerization reaction is analyzed based on the magnitude of the fluorescence intensity in the simulation experiment corresponding to each bacterial lysis buffer. Under the same polymerization reaction conditions, the higher the fluorescence signal intensity after the polymerization reaction, the higher the reaction efficiency of the polymerization reaction.

[0137] 3) The reversible terminator in step (2) is made fluorescent. In this step, under the action of bacterial lysis buffer, if the reversible terminator binds to the 3' end of the sequencing primer, the signal generated by the fluorescent group carried by the reversible terminator can be detected. After the polymerization reaction is completed, the fluorescence intensity of the nucleic acid complex on the surface of the solid support is detected, and the reaction efficiency of the polymerization reaction is analyzed based on the fluorescence intensity. Under the same polymerization reaction conditions, the higher the fluorescence signal intensity after the polymerization reaction, the higher the reaction efficiency of the polymerization reaction.

[0138] 4) After the polymerization reaction is complete, collect the sequencing primer chains. For example, the sequencing primer chains can be obtained by pre-setting restriction enzyme sites in the sequencing primers and then digesting the primers after the polymerization reaction. It should be noted that for simulated reversible terminator sequencing experiments performed on different bacterial lysates, the sequences of the sequencing primers and the restriction enzyme sites in the sequencing primers are completely identical. Of course, the methods for obtaining sequencing primer chains are not limited to the examples listed. Other methods for obtaining sequencing primer chains are also applicable to this invention. The sequencing primer chains are detected using capillary electrophoresis, and the amount of extended and non-extended sequencing primers in the sequencing primer chains is analyzed. The reaction efficiency of the polymerization reaction is analyzed based on the amount of extended sequencing primers. In some examples, the reaction efficiency of the polymerization reaction is analyzed based on the amount of extended sequencing primers, including: calculating the reaction efficiency of the polymerization reaction by dividing the amount of extended sequencing primers by (the amount of extended sequencing primers + the amount of non-extended sequencing primers).

[0139] In some embodiments of this application, the first detection step, in which the initial screening mutants are obtained by screening based on the detection results, further includes: based on the above detection results, screening to remove low-activity mutants, and the remaining mutants are the initial screening mutants.

[0140] In one implementation of this application, the first detection step further includes performing SDS-PAGE detection on the bacterial lysate to verify the protein expression levels of different mutant strains. The protein concentration in the bacterial lysate cannot be quantified using methods such as microplate readers; therefore, SDS-PAGE detection can roughly reveal the differences in expression levels between different mutants, thereby providing a rough estimate of the enzyme response during enzyme activity screening in the pre-screening phase.

[0141] In this embodiment of the application, the screening and culture step includes: placing the bacterial culture or single clone corresponding to the initial screening mutant into a bacterial culture medium for fermentation, adding a protein expression inducer during the fermentation process to induce mutant expression, and then extracting and purifying the mutant protein.

[0142] In this embodiment, the bacterial culture or single clone corresponding to the initially screened mutant is placed in a bacterial culture medium for fermentation. This method allows for batch cultivation of multiple mutant single clones, and also enables batch operation in the induction step, achieving high-throughput screening.

[0143] In one implementation of this application, the step of fermenting the bacterial culture or single clone corresponding to the initially screened mutant in a bacterial culture medium includes two fermentations: primary seed fermentation and secondary seed fermentation. Primary seed fermentation allows the initially screened mutant to rapidly multiply in a short time, obtaining a large number of clones; secondary seed fermentation increases fermentation density and seed vigor, ultimately improving the production efficiency of the mutant and ensuring its fermentation effect.

[0144] During secondary seed fermentation, a protein expression inducer can be added to induce mutant expression. In some embodiments, the protein expression inducer is selected from at least one of isopropyl-β-D-thiogalactoside and L-arabinose.

[0145] For example, the screening and culture step specifically includes the following operations:

[0146] 1. Primary seed fermentation: The pre-screened mutants that showed excellent performance were cultured in 10 mL LB medium under the following fermentation conditions: overnight culture at 37°C and 200 rpm.

[0147] 2. Secondary seed fermentation and induction of target protein expression: The fermented primary seeds were added to 10 mL of LB medium at an inoculation ratio of 1:100 and cultured at 37℃ and 200 rpm for 3 h. Then, 500 μM IPTG was added and the target protein expression was induced overnight at 25℃ and 200 rpm.

[0148] 3. Collection and lysis of bacteria: Centrifuge at 3500 rpm to collect the induced bacterial culture and discard the supernatant. Resuspend the bacteria in 1 mL of resuspension reagent containing lysis buffer. Incubate at room temperature with shaking at 20 rpm for 1 hour, until the bacterial culture becomes visibly clear. If the bacteria remain cloudy, extend the lysis time to allow the target protein to be lysed.

[0149] 4. His-Beads Protein Purification: Centrifuge the lysis buffer at maximum speed for 20 minutes using a desktop centrifuge until all the white precipitate settles to the bottom of the tube. If white flocculent matter remains suspended in the supernatant, extend the centrifugation time until all the white precipitate settles to the bottom of the tube. Collect the supernatant, add 1 mL of His-Beads, and incubate at 20 rpm at room temperature with shaking for 1 hour. After incubation, remove the magnetic beads using a magnetic rack and discard the supernatant. Add 1 mL of Ni Binding Buffer, pipette the magnetic beads 3-5 times, remove the magnetic beads using a magnetic rack, and discard the supernatant. Add 100 μL of Ni Elution Buffer, pipette the magnetic beads 3-5 times, remove the magnetic beads using a magnetic rack, and collect the supernatant. The supernatant is the protein purified by His-Beads, i.e., the purified mutant protein.

[0150] The second detection step includes: using mutant proteins to simulate reversible terminators for sequencing, and further screening the initially screened mutants based on the detection results to obtain sequencing enzyme mutants that meet the expected activity.

[0151] In one implementation of this application, before performing the second detection, the method further includes: using an enzyme-linked immunosorbent assay (ELISA) reader to detect the concentration of the mutant protein purified by His-Beads, and using SDS-PAGE to detect the purity of the purified protein to verify the expression level of different mutant proteins, and then performing simulated reversible terminator sequencing.

[0152] The simulated reversible terminator sequencing used in the second detection step can be the same as or different from the simulated reversible terminator sequencing used in the first detection step. In one implementation of this application, the sequencing reversible termination reaction includes: (1) providing a nucleic acid complex immobilized on the surface of a solid-phase support, the nucleic acid complex containing a nucleic acid template and sequencing primers whose sequences at least partially bind to the nucleic acid template; (2) adding a solution containing bacterial lysis buffer and a reversible terminator, and contacting the solution with the nucleic acid complex under polymerization reaction conditions; (3) detecting the reaction efficiency of each analyte in the polymerization reaction, and evaluating the activity of the analyte based on the reaction efficiency.

[0153] This application's embodiments simulate a reversible termination sequencing reaction in solution, which can completely replicate the real sequencing environment of the sequencing enzyme, in order to screen for sequencing enzyme mutants with better activity in real sequencing.

[0154] In step (1), the solid-phase support is understood as a carrier or support for immobilizing nucleic acid molecules or nucleic acid templates. In some application examples, the solid-phase support may also be referred to as a solid substrate, sequencing chip, or sequencing biochip. For example, the solid-phase support may be a substrate, magnetic beads, microspheres, or nanoparticles.

[0155] In this embodiment, a nucleic acid template is immobilized on the surface of a solid-phase support, and the nucleic acid template is attached to the surface of the solid-phase support. In some embodiments, a probe is attached to the surface of the solid-phase support, and the probe is covalently linked to a molecular / group on the surface of the solid-phase support, with the nucleic acid template attached to the surface of the solid-phase support via the probe. The probe is an oligonucleotide or nucleic acid molecule fragment capable of hybridizing with a target nucleic acid molecule of interest. In one embodiment, at least a portion of the probe is configured to hybridize with at least a portion of the 3' end of the nucleic acid template. In another embodiment, one end of the nucleic acid template is covalently linked to a molecular / group on the surface of the solid-phase support, thereby attaching to the surface of the solid-phase support. The nucleic acid template can further bind sequencing primers via base complementarity; in one embodiment, the probe acts as a sequencing primer, forming a nucleic acid complex with the nucleic acid template.

[0156] In step (2), the bacterial lysis buffer is derived from the lysis buffer obtained by lysing the bacterial cells in the pre-screening culture step, and it replaces the sequencing enzyme required for the sequencing reaction. The reversible terminators include reversible terminators dATP, dTTP, dGTP, and dCTP. Furthermore, the reaction solution used to simulate the pseudo-terminator sequencing also contains synthetic reagents, including but not limited to buffer salts and magnesium ions. In some embodiments, the solution also includes monovalent metal ions such as sodium or potassium ions and ammonium ions. It should be understood that in the embodiments of this application, the bacterial lysis buffer derived from each mutant clone is prepared with the pseudo-terminator solution and then contacted with the nucleic acid complex.

[0157] In this embodiment, the solution is contacted with the nucleic acid complex under suitable conditions for polymerization. Under these conditions, the nucleic acid complex, the lysing protein in the bacterial lysate, and the reversible terminator come into contact. However, when the bacterial lysate contains a polymerase-active protein that can promote the polymerization of the pseudoterminator with the nucleic acid complex, the pseudoterminator can bind to the nucleic acid complex and polymerize at the 3' end of the sequencing primer chain to extend the length by one nucleotide.

[0158] In some embodiments of this application, during simulated reversible terminator sequencing, a first reaction and a second reaction with different polymerization times are set, with the first reaction having a shorter reaction time than the second reaction. By setting two sets of reaction times, the differences in polymerization reactions at the two times can be compared, which is more beneficial for screening dominant strains. For example, given two mutants with similar polymerization activity under the second reaction condition, if the first mutant exhibits higher polymerization activity than the second mutant under the first reaction condition, then the first mutant is considered the dominant mutant compared to the second mutant.

[0159] In some embodiments, the time difference between the first reaction and the second reaction is greater than or equal to 1 minute. In some embodiments, the reaction time of the first reaction is 10-30 seconds, and the reaction time of the second reaction is 1.5-3 minutes. For example, the second reaction time is 2 minutes, and the first reaction time is 15 seconds. When the time difference between the first reaction and the second reaction is large, and the activity differences of various mutants are similar, further testing can be performed by setting reactions with time differences.

[0160] After the predetermined reaction time, the unreacted reversible terminator is removed by washing.

[0161] In step (3), the reaction efficiency of each analyte in the polymerization reaction is detected, and the activity of each bacterial lysate is evaluated based on the obtained reaction efficiency.

[0162] In some embodiments of this application, when a first reaction and a second reaction with different polymerization reaction times are set during simulated reversible terminator sequencing, and the reaction time of the first reaction is shorter than that of the second reaction, step (3) includes: comparing the reaction efficiency of the mutant protein under the first reaction condition and the reaction efficiency under the second reaction condition, analyzing the difference in reaction efficiency between the two reactions, and screening sequencing enzyme mutants based on the difference in reaction efficiency.

[0163] The reaction efficiency of polymerization of each analyte in this application embodiment can be detected by various methods. As an example, at least one of the following methods can be used:

[0164] 1) The sequencing primers in the nucleic acid complex of step (1) are made to carry fluorescent groups. In some examples, fluorescent groups are introduced at the 3' end of the sequencing primers. A fluorescence quenching group is introduced into the reversible terminator in step (2). In this step, under the action of bacterial lysis buffer, if the reversible terminator binds to the 3' end of the sequencing primer, the fluorescence quenching group carried by the reversible terminator will cause fluorescence quenching of the sequencing primer; if the reversible terminator does not bind to the 3' end of the sequencing primer, the signal generated by the fluorescent group carried by the sequencing primer can be detected before and after the polymerization reaction. In step (3), the change in fluorescence intensity before and after the polymerization reaction is detected, and the reaction efficiency of the polymerization reaction is analyzed based on the change in fluorescence intensity. Under the same polymerization reaction conditions, the greater the decrease in fluorescence signal intensity after the polymerization reaction relative to the fluorescence signal intensity before the polymerization reaction, the higher the reaction efficiency of the polymerization reaction.

[0165] 2) The sequencing primers in step (1) are made to carry a fluorescence quenching group. In some examples, a fluorescence quenching group is introduced at the 3' end of the sequencing primers. A fluorescent group is introduced into the reversible terminator in step (2). In this step, under the action of the bacterial lysis buffer, if the reversible terminator binds to the 3' end of the sequencing primer, the fluorescence quenching group carried by the sequencing primer will cause the fluorescence of the reversible terminator to be quenched; if the reversible terminator does not bind to the 3' end of the sequencing primer, the signal generated by the fluorescent group carried by the reversible terminator bound to the nucleic acid complex can be detected. In step (3), the fluorescence intensity after the polymerization reaction is detected, and the reaction efficiency of the polymerization reaction is analyzed based on the magnitude of the fluorescence intensity in the simulation experiment corresponding to each bacterial lysis buffer. Under the same polymerization reaction conditions, the higher the fluorescence signal intensity after the polymerization reaction, the higher the reaction efficiency of the polymerization reaction.

[0166] 3) The reversible terminator in step (2) is made fluorescent. In this step, under the action of bacterial lysis buffer, if the reversible terminator binds to the 3' end of the sequencing primer, the signal generated by the fluorescent group carried by the reversible terminator can be detected. After the polymerization reaction is completed, the fluorescence intensity of the nucleic acid complex on the surface of the solid support is detected, and the reaction efficiency of the polymerization reaction is analyzed based on the fluorescence intensity. Under the same polymerization reaction conditions, the higher the fluorescence signal intensity after the polymerization reaction, the higher the reaction efficiency of the polymerization reaction.

[0167] 4) After the polymerization reaction is complete, collect the sequencing primer chains. For example, the sequencing primer chains can be obtained by pre-setting restriction enzyme sites in the sequencing primers and then digesting the primers after the polymerization reaction. It should be noted that for simulated reversible terminator sequencing experiments performed on different bacterial lysates, the sequences of the sequencing primers and the restriction enzyme sites in the sequencing primers are completely identical. Of course, the methods for obtaining sequencing primer chains are not limited to the examples listed. Other methods for obtaining sequencing primer chains are also applicable to this invention. The sequencing primer chains are detected using capillary electrophoresis, and the amount of extended and non-extended sequencing primers in the sequencing primer chains is analyzed. The reaction efficiency of the polymerization reaction is analyzed based on the amount of extended sequencing primers. In some examples, the reaction efficiency of the polymerization reaction is analyzed based on the amount of extended sequencing primers, including: calculating the reaction efficiency of the polymerization reaction by dividing the amount of extended sequencing primers by (the amount of extended sequencing primers + the amount of non-extended sequencing primers).

[0168] In this embodiment, the principle of capillary electrophoresis (CE) is as follows: Capillary electrophoresis uses a high-voltage electric field as the driving force and a capillary as the separation channel. The capillary is filled with buffer or gel, and separation is achieved based on the differences in mobility and distribution among the components in the sample. The high-voltage electric field can drive separation through different mass-to-charge ratios of molecules; the gel acts as a molecular sieve, separating molecules based on their different molecular weights. In some embodiments, the reaction efficiency of sequencing enzymes is assessed using a combination of magnetic beads and capillary electrophoresis. For example, after a reversible termination extension reaction simulating sequencing is performed on the magnetic beads, the mixture is denatured with 50% formamide. The 50% formamide solution contains: the sequencing products that have undergone extension (primers with nucleotide extension) and the unreacted, unextended sequencing primers (primers without extension). By labeling the 5' end of the sequencing primers with FAM, the fluorescence absorption peaks of the extended sequencing primers (primers with nucleotide extension) and the unreacted primers (primers without extension) can be observed on capillary electrophoresis. Since the unreacted primers (primers without extension) and the products (primers with nucleotide extension) differ by a reversible termination base, a POP6 gel with appropriate resolution can be used to observe the peak difference on capillary electrophoresis. Integrating the areas of the unreacted primer peaks (primers without extension) and the product peaks (primers with nucleotide extension) yields the amount of unreacted primers (i.e., the amount of unextended sequencing primers) and the amount of product (primers with nucleotide extension) (i.e., the amount of extended sequencing primers). The reaction efficiency of the analyte (i.e., bacterial lysate or mutant protein) with the substrate is calculated by dividing the product amount by (product amount + amount of unextended sequencing primers). The activity of the analyte is then evaluated based on this reaction efficiency.

[0169] This application establishes a screening system combining "pre-screening" and "screening," which allows for the initial screening process to eliminate monoclonal mutants with random mutations that reduce function, while mutants with significantly enhanced function are used as initial screening mutants and proceed to the next screening stage for more refined selection. This "pre-screening" approach, combined with simulated reversible terminator sequencing, can efficiently screen out inferior mutants and allow for multiple screening of superior mutants under restrictive conditions, ultimately yielding the best sequencing enzyme mutants efficiently and in high throughput.

[0170] This application uses the above screening method to screen a series of 9°N DNA polymerase mutants and obtains mutants with at least good DNA synthesis activity, namely the DNA polymerase of this application.

[0171] The following example uses DNA polymerase number 54 (compared to 9°N DNA polymerase (UniProt ID: Q56366; PDB ID: 5OMQ), which contains the following mutation sites: D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G) from 9°N. Based on DNA polymerase mutants, mutants or combinations of mutants containing the E734R / K, G677R / K, A675R / K, and P739R / K mutation sites are designed. Examples of combinations of the E734R / K, G677R / K, A675R / K, and P739R / K mutation sites include: L631M+E734R / K, G677R / K+A675R / K+E734R / K, A675R / K+E734R / K, and E734R / K+P739R / K. These are used as examples for illustration.

[0172] Example 1

[0173] Design the following mutation sites and their combinations:

[0174] E734R / K (No. 94 / No. 194), G677R / K (No. 130 / No. 230), A675R / K (No. 131 / No. 231), P739K / R (No. 206 / No. 306), L631M+E734R (No. 170), L631M+E734K (No. 270), G677R+A675R+E734R (No. 179), G677R+E734R (No. 203), A675R+E734R (No. 204), A675K+E734R (No. 304), E734R+P739R (No. 207), E734R+P739K (No. 307).

[0175] The mutant protein containing the above-mentioned numbers was used to simulate "reversible terminator sequencing" in solution. The experimental protocol is as follows: Figure 1 Specifically, it includes:

[0176] 1) Primer Annealing:

[0177] The sequence shown in Seq ID No. 1 was designed as a nucleic acid template, which has a 3' end biotin modification.

[0178] Seq ID No.1:

[0179] 5'-AATGATACGGCGACCACCGAGATCTAGATGATTTCATTCTTGTTATTT ATAACTCTTATATTG-Biotin-3'

[0180] The sequencing primers were the sequence shown in Seq ID No. 2, with a 5' FAM modification, which forms the basis for capillary electrophoresis (CE) in the subsequent examples. The sequencing primers were reverse complementary and bound to the 3' end of the template strand.

[0181] Seq ID No. 2:

[0182] 5'-FAM-CAATATAAGAGTTATAAATAACAAGAATGAAATC-3'

[0183] The nucleic acid template and sequencing primers were annealed. The annealing reaction system consisted of 20 μL of 10 mmol / L nucleic acid template, 20 μL of 10 mmol / L sequencing primers, and 160 μL of ddH2O. The reaction conditions were as follows: hot cap temperature 105℃, 94℃ for 2 min, 65℃ for 30 s, 60℃ for 30 s, 55℃ for 30 s, 50℃ for 30 s, 42℃ for 30 s, and standby temperature 25℃.

[0184] Once the reaction is complete, the annealed product is obtained.

[0185] 2) Dynabeads-Primer Annealing Coupling

[0186] Dynabeads are coupled with a streptomycin layer that can be coupled to a 3' end biotin-modified template strand, thereby linking the template-primer annealing product to magnetic beads, such as... Figure 5 As shown.

[0187] Coupling reaction system: 125 μL of annealed product, 500 μL of 2×BW magnetic beads, and 375 μL of 1×TE.

[0188] Incubate at room temperature, invert for 15 minutes, then adsorb using a magnetic rack and discard the supernatant. Wash three times with 1×TE buffer, and finally place in 1×TE buffer for later use; this is the nucleic acid complex immobilized on the surface of the solid support.

[0189] 3) Simulated reversible sequencing termination (Dynabeads Solution Reaction)

[0190] Nucleic acid complexes immobilized on magnetic beads, reaction substrates (modified dNTPs), a reversible terminator (containing a fluorescent label), and the aforementioned mutant single clone numbered 54 were provided. The mixture was incubated in a PCR instrument at 55°C for 30 seconds, followed immediately by the addition of EDTA to terminate the reaction. The magnetic beads were adsorbed using a magnetic rack, and the supernatant was removed. The mixture was washed three times with 1×TE, and denatured with 50% formamide to open the double strands, causing both extended and non-extended sequencing primers to fall off. The supernatant was used for subsequent experimental detection. The reversible termination synthesis reaction system consisted of: 5 μL of magnetic beads with surface-immobilized nucleic acid complexes, 2.25 μL of sequencing enzyme (i.e., mutant protein, 2 mg / mL), and 42.75 μL of sequencing buffer without sequencing enzyme. Reaction conditions: PCR instrument incubation at 55°C for 30 seconds, followed immediately by the addition of EDTA to terminate the reaction.

[0191] To verify the enzymatic activity of mutant proteins in different reaction systems, two parallel experiments with different reaction times were set up for each mutant protein. Specifically, the mutant proteins were subjected to reversible terminator sequencing in both a Slow-Reaction and Fast-Reaction system. The difference in reaction efficiency between the slow and fast reaction conditions was analyzed and compared to screen for mutant proteins with superior activity. Specifically, the reaction conditions for the slow-reaction screening system were: incubation at 55°C for 30 seconds in a PCR instrument, followed immediately by the addition of EDTA to terminate the reaction. The reaction conditions for the fast-reaction screening system were: incubation at 55°C for 15 seconds in a PCR instrument, followed immediately by the addition of EDTA to terminate the reaction.

[0192] The relative activity of mutant proteins was characterized by statistically analyzing the fluorescence intensity of different mutants. Specifically, five parallel experiments were conducted for each mutant protein, and corresponding fluorescence data were collected. After removing the maximum and minimum values, background noise was subtracted. The fluorescence values ​​of the experimental groups were standardized and compared using the WT (Wavelength Tolerance) as the background. The standardized value was calculated as follows: Standardized fluorescence value = (Mutant fluorescence intensity - Background fluorescence intensity) ÷ (WT fluorescence intensity - Background fluorescence intensity) × 100%.

[0193] The fluorescence data of each mutant protein obtained in Slow-Reaction and Fast-Reaction are statistically analyzed as follows. Figure 2and Figure 3 As shown, where, Figure 2 The graph shows the fluorescence data of WT and numbers 94, 130, 131, 170, 179, 203, 204, 206, and 207. Figure 3 Statistical graph of fluorescence data for WT and numbers 194, 230, 231, 270, 304, 306, and 307.

[0194] The results showed that, regardless of whether it was a fast or slow reaction, the mutants or combinations numbered 94, 130, 131, 170, 179, 203, 204, 206, and 207 all exhibited enhanced fluorescence intensity after the reaction compared to the control group (WT). Particularly in the fast-reaction experiment with shorter reaction times, the mutants or combinations numbered 94, 130, 131, 170, 179, 203, 204, 206, and 207 showed a more significant advantage, especially the mutants or combinations numbered 94, 131, 204, 207, 194, 231, 306, and 307, which showed more pronounced polymerase activity. It is noteworthy that the reaction time of the fast reaction was also closer to the actual polymerization reaction time in sequencing, further highlighting the differences in polymerase activity among the mutants.

[0195] Example 2

[0196] The mutant protein provided in Example 1 was subjected to "reversible terminator sequencing" in solution using the same method as in Example 1.

[0197] After completing the reversible termination extension reaction, the denatured products were denatured with 50% formamide and subjected to capillary electrophoresis (using POP6 gel). Due to the FAM label at the 5' end of the sequencing primers, the denatured products, including the extended sequencing primers and the unreacted primers (i.e., non-extended primers), can be observed on capillary electrophoresis, and their fluorescence absorption peaks are detected. Since the extended sequencing primers and the unreacted primers differ in molecular weight by one nucleotide, the peak difference between them can be observed by capillary electrophoresis. By integrating the areas of the unreacted primer peak (primer without extension) and the product peak (primers with nucleotide extension), the amount of unreacted primer (i.e., the amount of non-extended sequencing primers) and the amount of product (primers with nucleotide extension) (i.e., the amount of extended sequencing primers) can be obtained. The reaction efficiency of the analyte (i.e., the mutant protein) with the substrate is calculated by dividing the product amount by (product amount + amount of non-extended sequencing primers).

[0198] The relative reaction efficiency of each mutant protein was statistically analyzed. The calculation formula is: relative reaction efficiency = reaction efficiency of mutant to substrate ÷ reaction efficiency of WT to substrate × 100%. The results are shown in Table 1.

[0199] Table 1. Test results for Slow-Reaction and Fast-Reaction

[0200]

[0201]

[0202] As shown in Table 1, compared to the polymerization efficiency of the control group WT, the reaction efficiencies of the mutants or combinations corresponding to numbers 94, 130, 131, 170, 179, 203, 204, 206, and 207 were enhanced in both Slow-Reaction and Fast-Reaction experiments, especially in the Fast-Reaction experiment, where the improvement in reaction efficiency was more significant. Among them, the mutants or combinations corresponding to numbers 94, 131, 204, 207, 194, 231, 306, and 307 showed significantly improved polymerization efficiency, which is consistent with the experimental results obtained in Example 1.

[0203] Example 3

[0204] Eight mutants, numbered 94, 131, 204, 207, 194, 231, 304, and 307, were selected and sequenced using the same conditions as in Example 1 (sequencing on a sequencing platform). Sequencing Q30, phasing, pre-phasing, and mismatch rate were statistically analyzed. Here, Q represents the sequencing quality parameter, and Q30 is an important quality control indicator used to measure the accuracy of sequencing data. Specifically, Q30 represents the proportion of bases with a Phred score greater than or equal to 30 during sequencing. According to the Phred quality scoring system, the error rate corresponding to Q30 is 0.1%, meaning that the number of incorrect bases per 1000 bases does not exceed one. This means that a higher Q30 value indicates higher reliability and a lower error rate in the sequencing data. The mismatch rate represents the percentage of bases incorrectly identified relative to the reference sequence in the sequencing results. A lower mismatch rate indicates higher sequencing accuracy and better sequencing quality.

[0205] Using the indicators of WT as standard values, the indicator values ​​of the remaining mutants were standardized (standardization formula = mutant indicator value ÷ WT indicator value × 100%). The differences in indicators between the mutants and WT were then compared, and the results are shown in Table 2. Statistical results are plotted as follows. Figure 4 and Figure 5As shown, where, Figure 4 The graph shows the fluorescence data statistics for WT and numbers 94, 131, 204, 207, 194, 231, 304, and 307. Figure 5 Statistical graph of fluorescence data for WT and numbers 94, 131, 204, 207, 194, 231, 304, and 307.

[0206] Table 2. Results of mutant sequencing data analysis

[0207]

[0208]

[0209] Figure 4 , Figure 5 The results in Table 2 show that, compared with the control group WT, mutants or mutant combinations numbered 94, 131, 204, 207, 194, 231, 304, and 307 generally exhibited better performance. Specifically, the Q30 index of mutants or mutant combinations numbered 94, 131, 204, 207, 194, 231, 304, and 307 was comparable to that of WT, especially the Q30 of mutants or mutant combinations numbered 204 and 304, which was significantly better than that of WT. The phasing index of mutants or mutant combinations numbered 94, 194, 304, and 204 was comparable to that of WT, while the phasing index of the remaining mutants or mutant combinations was significantly lower than that of WT. The pre-phasing index of mutants or mutant combinations numbered 94 and 194 was significantly lower than that of WT. The ng index was comparable to that of WT, while the pre-phasing index of the remaining mutants or mutant combinations was better than that of WT, especially the pre-phasing of mutants or mutant combinations numbered 131, 231, and 307, which was significantly lower than that of WT. Compared with the control group WT, the mismatch rate of mutants or mutant combinations numbered 94, 131, 204, 207, 194, 231, 304, and 307 was lower, especially the mismatch rate of mutants or mutant combinations numbered 94, 207, 194, and 307 was significantly lower.

[0210] In summary, the E734R mutant can significantly reduce the error rate and improve sequencing accuracy; the A675R mutant can significantly reduce phasing and pre-phasing during sequencing, thus improving the overall sequencing process. The combined mutant GM204 can significantly improve the Q value during sequencing.

[0211] To analyze the reasons for the above results, the crystal structures of some proteins of the 9°N polymerase (UniProt ID: Q56366; PDBID: 5OMQ) and some mutant sites of the 9°N polymerase were further analyzed under X-ray diffraction conditions. The results are as follows: Figure 6 , Figure 7 As shown.

[0212] Depend on Figure 6 It is evident that the A675 site plays an auxiliary role in the binding of sequencing enzyme and double-stranded DNA template; mutations at this site can affect the interaction between the DNA template and the sequencing enzyme. The G677 site, spatially parallel and adjacent to the A675 site, also plays an auxiliary role in the binding of sequencing enzyme and double-stranded DNA template; mutations at this site can affect the interaction between the sequencing enzyme and the DNA template. The E734 site is located on an α-helix parallel to the double-stranded DNA template; this region plays an important role in the accurate insertion of the DNA template and subsequent base binding; mutations at this site affect the sequencing activity of the sequencing enzyme. The P739 site, located on the same α-helix secondary structure, is also parallel to the double-stranded DNA template; this region plays an important role in DNA template binding and the stability of the enzyme-DNA complex.

[0213] Depend on Figure 7 It can be seen that the 'Positive charged dimer align with DNA template' formed by E734R+R743 and the phosphate group of the dsDNA template, that is, the divalent positive charge formed by the positive charge of the two sites, and spatially aligned with the DNA template height; the 'Positive charged trimer align with DNA template' formed by E734R+P739R+R743 and the phosphate group of the dsDNA template, that is, the trivalent positive charge formed by the positive charge of the three sites, and spatially aligned with the DNA template height.

[0214] Analysis suggests that the E734R / K mutation enhances the binding of sequencing enzymes to the DNA template and stabilizes the accurate embedding of the DNA template, thereby improving sequencing accuracy and efficiency. Mutating Glu at position 734 to Arg / Lys increases both the interaction distance between the side chain residues and the DNA template, and the charge interaction between the side chain residues and the phosphate groups of the DNA template, thus enhancing the binding ability of the sequencing enzyme to the DNA template. The α-helix at position E734 exhibits high spatial alignment with the DNA template, such as... Figure 6As shown, after the E734 mutation to Arg / Lys, together with the original Arg at position 743, they can form a 'Positive charged dimer align with DNA template', that is, the positive charges of the two sites form a divalent positive charge interaction, which is spatially aligned with the DNA template, thereby strengthening the interaction between the sequencing enzyme and the DNA template, as shown. Figure 7 As shown.

[0215] The P739K / R site is also located on an α-helix highly aligned with the DNA template. Changing the Pro at site 739 to Arg / Lys stabilizes the sequencing enzyme-DNA complex through charge interaction, thereby enhancing sequencing quality. On the other hand, the P739K / R and E734K / R mutations, together with the original E743, form a 'Positive charged trimer align with DNA template', meaning a trivalent positive charge interaction formed by the positive charges at the three sites, spatially aligned with the DNA template. Figure 7 As shown in the diagram. This structure greatly stabilizes the binding of sequencing enzymes and DNA templates, specifically resulting in reduced phasing and increased Q values ​​in subsequent sequencing.

[0216] The G677R and A675R sites are located on a loop embedded within the DNA groove. Mutations at these sites can enhance the stability of the sequencing enzyme-DNA complex, thereby increasing the activity of the sequencing enzyme. Mutating Gly at position 677 and Ala at position 675 to Arg causes the loop region embedded in the DNA groove to bind more stably to the DNA backbone phosphate groups through charge interactions, thus improving the stability of the sequencing enzyme-DNA complex. Figure 7 As shown.

[0217] The information presented by the protein crystal structure can, to some extent, explain the test results obtained in Examples 1 to 3.

[0218] Example 4

[0219] A method for screening monoclonal mutants of the above is provided, including pre-screening culture:

[0220] 1) Transformation: The mutant clone was transformed, and BL21 DE3 competent cells were cultured after being transformed with 100 ng of plasmid.

[0221] 2) Primary seed fermentation: On the second day, select single clones and add them to a 96-well plate to culture the mutant bacterial culture, with 200 μL of bacterial culture in each well. Culture the primary seed overnight at 37°C and 200 rpm. This step requires sealing the 96-well plate with sealing film to prevent liquid evaporation.

[0222] 3) Secondary seed fermentation and induction of target protein expression: The fermented primary seed was added to LB medium in 96-well plates at an inoculation ratio of 1:100 (200 μL per well) and cultured at 37°C and 200 rpm for 3 h. Then, 500 μM IPTG (isopropyl-β-D-thiogalactoside) was added, and the target protein expression was induced overnight at 25°C and 200 rpm.

[0223] 4) Bacterial lysis and protein efflux: Add lysis buffer to the bacterial culture in a 1:10 ratio in a 96-well plate, and incubate at room temperature with shaking at 20 rpm for 1 hour to lyse the target protein and obtain the bacterial lysis buffer.

[0224] The first detection step, namely, detecting the bacterial lysate obtained in step 4), specifically includes:

[0225] The bacterial lysate was subjected to SDS-PAGE to detect and statistically analyze protein expression levels. In this embodiment, the SDS-PAGE protein expression level statistical method is as follows: The SDS-PAGE image after electrophoresis, staining, and destaining was scanned and loaded into ImageJ. Image>Type>8bit (click Image, Type, 8bit in sequence) to convert the image to 8-bit resolution format; Process>SubtractBackground (click Process and Subtract Background in sequence), adjusting parameters until the target band is visible and the background noise is minimized. Select [Rectangle Selection], select the target band whose grayscale value you want to analyze, press "Ctrl+1", then select the target bands whose grayscale value you want to analyze further, press "Ctrl+2", until all the target bands are selected, then press "Ctrl+3". A grayscale peak will appear. Select the [Magic Wand] tool to close the integral area. After all selections are complete, press K, and the integral area statistical value will appear. This statistical value is the grayscale value of the target protein band, and this grayscale value is used to characterize the amount of the target protein band.

[0226] The results showed that all mutant monoclonal strains and bacteria numbered 54 could effectively induce protein expression.

[0227] Simulate "reversible terminator sequencing" in solution; experimental protocol referenced. Figure 1 Specifically, it includes:

[0228] 1) Primer Annealing:

[0229] The sequence shown in Seq ID No. 1 was designed as the nucleic acid template, with a 3' biotin modification. The sequencing primers were the sequence shown in Seq ID No. 2, with a 5' FAM modification, to prepare for the next step of capillary electrophoresis (CE). The sequencing primers were reverse complementary and bound to the 3' end of the template strand. Seq ID No. 1 and Seq ID No. 2 were the same as in Example 1.

[0230] The nucleic acid template and sequencing primers were annealed. The annealing reaction system consisted of 20 μL of 10 mmol / L nucleic acid template, 20 μL of 10 mmol / L sequencing primers, and 160 μL of ddH2O. The reaction conditions were as follows: hot cap temperature 105℃, 94℃ for 2 min, 65℃ for 30 s, 60℃ for 30 s, 55℃ for 30 s, 50℃ for 30 s, 42℃ for 30 s, and standby temperature 25℃.

[0231] Once the reaction is complete, the annealed product is obtained.

[0232] 2) Dynabeads-Primer Annealing Coupling

[0233] Dynabeads are coupled with a streptomycin layer that can be coupled to a 3' end biotin-modified template strand, thereby linking the template-primer annealing product to magnetic beads, such as... Figure 1 As shown.

[0234] Coupling reaction system: 125 μL of annealed product, 500 μL of 2×BW magnetic beads, and 375 μL of 1×TE.

[0235] Incubate at room temperature, invert for 15 minutes, then adsorb using a magnetic rack and discard the supernatant. Wash three times with 1×TE buffer, and finally place in 1×TE buffer for later use; this is the nucleic acid complex immobilized on the surface of the solid support.

[0236] 3) Simulated reversible sequencing termination (Dynabeads Solution Reaction)

[0237] Nucleic acid complexes immobilized on magnetic beads, reaction substrates (modified dNTPs), a reversible terminator (containing a fluorescent label), and lysate of a mutant single clone and bacterial culture (number 54) were provided. The mixture was incubated in a PCR instrument at 55°C for 30 seconds, followed immediately by the addition of EDTA to terminate the reaction. The magnetic beads were adsorbed using a magnetic rack, and the supernatant was removed. The mixture was washed three times with 1×TE, and denatured with 50% formamide to open the double strands, causing both extended and non-extended sequencing primers to fall off. The supernatant was used for subsequent experimental detection. The reversible termination synthesis reaction system consisted of: 5 μL of magnetic beads with surface-immobilized nucleic acid complexes, 2.25 μL of sequencing enzyme (i.e., bacterial culture lysate or mutant protein), and 42.75 μL of sequencing buffer without sequencing enzyme. The reaction conditions were: incubation in a PCR instrument at 55°C for 30 seconds, followed immediately by the addition of EDTA to terminate the reaction. The relative activity of the mutant protein was then characterized by statistical analysis of the fluorescence intensity of different mutants.

[0238] The initial screening results showed that the sequencing enzyme activity of some irrational mutants was lower than that of the control group (WT 54), indicating reduced activity in these mutants. These mutants with reduced activity were removed during the initial screening process, and the remaining mutants with higher activity, i.e., the initial screening mutants, proceeded to the next screening experiment. Specifically, this included:

[0239] 1) Primary seed fermentation: The pre-screened mutants that showed excellent performance were cultured in 10 mL LB medium. The fermentation conditions were 37℃ and 200 rpm overnight.

[0240] 2) Secondary seed fermentation and induction of target protein expression: The fermented primary seeds were added to 10 mL of LB medium at an inoculation ratio of 1:100 and cultured at 37℃ and 200 rpm for 3 h. Then, 500 μM IPTG was added and the target protein expression was induced overnight at 25℃ and 200 rpm.

[0241] 3) Bacterial collection and lysis: Centrifuge at 3500 rpm to collect the induced bacterial culture and discard the supernatant. Resuspend the bacterial cells in 1 mL of resuspension reagent containing lysis buffer. Incubate at room temperature with shaking at 20 rpm for 1 hour, until the bacterial culture becomes visibly clear. If the bacterial culture remains cloudy, extend the lysis time to allow the target protein to be lysed.

[0242] 4) His-Beads Protein Purification: Centrifuge the lysis buffer at maximum speed for 20 minutes using a desktop centrifuge until all the white precipitate settles to the bottom of the tube. If white flocculent matter remains suspended in the supernatant, extend the centrifugation time until all the white precipitate settles to the bottom of the tube. Collect the supernatant, add 1 mL of His-Beads, and incubate at room temperature with shaking at 20 rpm for 1 hour. After incubation, use a magnetic rack to remove the magnetic beads and discard the supernatant. Add 1 mL of Ni Binding Buffer, pipette the magnetic beads 3-5 times, and then use a magnetic rack to remove the magnetic beads and discard the supernatant. Add 100 μL of Ni Elution Buffer, pipette the magnetic beads 3-5 times, and then use a magnetic rack to remove the magnetic beads and collect the supernatant. The supernatant is the protein purified by His-Beads, i.e., the purified mutant protein.

[0243] The purified mutant proteins were analyzed by SDS-PAGE to determine protein quantity and by microplate reader to measure concentration, and sequenced using a simulated reversible terminator. Results showed that monoclonal strains of each initially screened mutant could effectively induce expression of the mutant protein.

[0244] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A DNA polymerase, characterized in that, The DNA polymerase has an amino acid sequence that is at least 80% identical to the amino acid sequence of the 9°N DNA polymerase, and the DNA polymerase includes at least one of the following first-class amino acid substitution mutations at positions A675, G677, E734, and P739 or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme.

2. The DNA polymerase according to claim 1, characterized in that, The first type of amino acid substitution mutation includes at least one of the substitution mutations of A675R, A675K, G677R, G677K, E734K, E734R, P739K and P739R or functionally equivalent substitution mutations; Optionally, the first type of amino acid substitution mutation includes one of the following mutations: (a)A675R / K; (b)G677R / K; (c)E734R / K; (d)P739R / K; (e) Combination mutations of L631M and E734R / K; (f) Combinatorial mutations of G677R / K, A675R / K and E734R / K; (g)A675R / K and E734R / K combined mutations; (h) Combination mutations of G677R / K and E734R / K; (i) Combined mutations of E734R / K and P739R / K.

3. The DNA polymerase according to claim 1 or 2, characterized in that, The DNA polymerase further includes at least one type II amino acid substitution mutation at the D141, E143, C223, L408, Y409, P410, L478, A485, Y497 and H633 positions or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme. Optionally, the second type of amino acid substitution mutation includes at least one of the following substitution mutations: D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, and H633G, or functionally equivalent substitution mutations. Optionally, the second type of amino acid substitution mutation includes one of the following mutations: (a) Combination mutations of D141A, C223S and Y497G; (b) Combination mutations of D141A, L408A, Y409A, P410I and H633G; (c) Combination mutations of D141A, L408A, Y409A, P410I and H633G; (d) Combination mutations of L408A, Y409A, P410I, L478S, A485L and H633G; (e) Combination mutations of D141A, C223S, L408A, Y409A, L478S, A485L and H633G; (f) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G and H633G.

4. The DNA polymerase according to any one of claims 1-3, characterized in that, The DNA polymerase also includes at least one type III amino acid substitution mutation at the T622 or L631 position or a functionally equivalent position on the 9°N amino acid sequence of the sequencing enzyme. Optionally, the third type of amino acid substitution mutation includes at least one of T622A, L631M substitution mutations or functionally equivalent substitution mutations; Optionally, the third type of amino acid substitution mutation includes a combined mutation of T622A and L631M; Optionally, the DNA polymerase further includes at least one of the following amino acid substitution mutations at or functionally equivalent positions on the 9°N amino acid sequence of the sequencing enzyme: M129, M329, P410, E576, T590, T590, H633, K705, I521, V278, K559, E599, L408, R247, T349, E580, S407, and V471. Optionally, the fourth type of amino acid substitution mutation includes at least one of the following: M129A, M329H, P410L, E576L, T590K, T590R, H633T, K705A, I521L, V278L, K559G, E599D, L408S, R247Y, T349R, E580K, S407A, and V471G substitution mutations or functionally equivalent substitution mutations. Optionally, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme: (a) Combination mutations of L631M, E734R and I521L; (b) Combination mutations of D141A, L408A, Y409A, P410I, H633G, L631M, E734R and I521L; (c) Combination mutations of D141A, C223S, L631M, E734R and I521L; (d) Combination mutations of D141A, L408A, Y409A, P410I, C223S, L631M, E734R and I521L; (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, L631M, E734R and I521L; (f) Combination mutations of V278L, L631M, E734R and I521L; (g) Combination mutations of L408A, Y409A, P410I, V278L, L631M, E734R and I521L; (h) Combination mutations of L408A, Y409A, P410I, L478S, A485L, V278L, L631M, E734R and I521L; (i) Combination mutations of D141A, L408A, Y409A, P410I, L478S, A485L, H633G, V278L, L631M, E734R and I521L; (j) Combinatorial mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, L631M, E734R and I521L; (k)L631M, E734R, I521L and E599D combined mutations; (l) Combination mutations of L408A, Y409A, P410I, L631M, E734R, I521L and E599D; (m) Combination mutations of E143A, C223S, L408A, Y409A, A485L, Y497G, L631M, E734R, I521L and E599D; (n) Combination mutations of D141A, E143A, C223S, L408A, Y409A, A485L, Y497G, L631M, E734R, I521L and E599D; (o) Combination mutations of E143A, C223S, L408A, Y409A, A485L, Y497G, L631M, E734R, I521L and E599D; (p) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, L631M, E734R, I521L and E599D; (q) Combination mutations of D141A, C223S, L408A, Y409A, A485L, L631M, E734R, I521L and E599D; (r) Combination mutations of L408A, Y409A, P410I, L631M, E734R, I521L and E599D; (s) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, L631M, E734R, I521L and E599D; Optionally, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme: (a) Combined mutations of K559G and E580K; (b) Combination mutations of L408A, Y409A, P410I, L478S, A485L, Y497G, K559G and E580K; (c) Combination mutations of D141A, L408A, Y409A, P410I, A485L, K559G and E580K; (d) Combination mutations of L408A, Y409A, P410I, K559G and E580K; (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, K559G and E580K; Optionally, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme: (a) Combined mutations of E734R and I521L; (b) Combination mutations of E143A, C223S, L408A, Y409A, P410I, A485L, Y497G, E734R and I521L; (c) Combination mutations of D141A, L408A, Y409A, P410I, A485L, E734R and I521L; (d) Combination mutations of L408A, Y409A, P410I, E734R and I521L; (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, E734R and I521L; Optionally, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme: (a) Combination mutations of E734R, I521L and E599D; (b) Combination mutations of L408A, Y409A, P410I, A485L, Y497G, H633G, E734R, I521L and E599D; (c) Combination mutations of D141A, E143A, C223S, L408A, Y409A, A485L, E734R, I521L and E599D; (d) Combination mutations of L408A, Y409A, P410I, E734R, I521L and E599D; (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, E734R, I521L and E599D; Optionally, the DNA polymerase includes one of the following mutations in the 9°N amino acid sequence of the sequencing enzyme: (a) Combination mutations of A675R, S407A and V471G; (b) Combination mutations of C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, A675R, S407A and V471G; (c) Combination mutations of E143A, C223S, L408A, Y409A, P410I, L478S, A675R, S407A and V471G; (d) Combination mutations of L408A, Y409A, P410I, L478S, A675R, S407A and V471G; (e) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G, V278L, A675R, S407A and V471G; (f) Combination mutations of D141A, E143A, C223S, L408A, Y409A, P410I, L478S, A485L, Y497G, H633G and E734R.

5. The DNA polymerase according to any one of claims 1-4, characterized in that, The DNA polymerase is a group B DNA polymerase; Optionally, the polymerase is selected from group B archaea DNA polymerase, human DNA polymerase-a, T4 polymerase, RB69 polymerase or phi29 phage DNA polymerase; Optionally, the group B archaea DNA polymerase is selected from the genera *Thermococcus*, *Firecoccus*, or *Methanococcus*.

6. A nucleic acid molecule, characterized in that, The DNA polymerase is encoded as described in any one of claims 1-5.

7. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 6.

8. A host cell, characterized in that, It includes the expression vector as described in claim 7.

9. The application of the DNA polymerase according to any one of claims 1-5 in nucleic acid sequencing.

10. A sequencing kit containing the DNA polymerase according to any one of claims 1-5; Optionally, the kit includes nucleotides; Optionally, the nucleotide contains a reversible terminator; Optionally, the reversible terminator comprises a purine or pyrimidine base and a ribose or deoxyribose moiety having a removable 3'-OH blocking group covalently linked thereto.

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