DNA polymerase mutant and use thereof in sequencing

Abstract

A DNA polymerase mutant and use thereof in sequencing. The DNA polymerase mutant comprises: compared to a Pyrococcus sp. GB-D DNA polymerase exo-mutant, the DNA polymerase mutant has at least three amino acid mutations at the following four sites or functionally equivalent sites: position 409, position 410, position 411, and position 486; the Pyrococcus sp. GB-D DNA polymerase exo-mutant has the amino acid sequence shown in SEQ ID NO: 1.

Description

DNA polymerase mutants and their applications in sequencing Technical Field

[0001] The present application relates to the field of gene sequencing. Specifically, the present application relates to DNA polymerase mutants and their application in sequencing. Background Art

[0002] Decoding nucleic acid sequences is crucial for understanding gene function and control mechanisms, as well as for numerous fundamental techniques in molecular biology. DNA sequencing plays a key role in this regard, and second-generation high-throughput DNA sequencing technology based on sequencing by synthesis (SBS) is currently the most widely used. Compared to first-generation sequencing methods, second-generation sequencing offers several significant advantages.

[0003] First, second-generation sequencing technology has the characteristics of fast sequencing speed and high throughput, which enables it to complete large-scale genome sequencing in a short period of time. Second, the relatively low sequencing cost has significantly reduced the sequencing price per base, making large-scale sequencing projects more economical and feasible. In addition, second-generation sequencing technology has demonstrated excellent sequencing accuracy and can detect low-frequency variants and heterozygous sites, which is of great significance for biological research and clinical diagnosis. More importantly, the high flexibility of this technology makes it suitable for different target regions and sample types, expanding its application areas, including transcriptome sequencing, epigenetic sequencing, whole-genome association analysis, drug resistance detection, genetic identification, forensic analysis, genetic counseling and medical diagnosis.

[0004] Sequencing polymerase plays a key role in the second-generation sequencing process. The core principle of second-generation sequencing is sequencing by synthesis, that is, determining the DNA sequence by capturing the special labels (usually fluorescent molecular labels) carried by the newly added bases during DNA replication. The task of sequencing polymerase is to pair and connect the labeled bases with the template chain to form a complementary DNA chain. This step is crucial for the success of sequencing. However, natural polymerases generally have low polymerization ability for artificially modified nucleotides or nucleotide analogs, which limits the efficiency and accuracy of sequencing.

[0005] To this end, DNA polymerases used for sequencing still need to be improved.

[0006] Summary of the Invention

[0007] The present application aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0008] To address the problems with polymerization speed and sequencing quality (e.g., lag, strand bias, etc.) of existing sequencing enzymes, the inventors engineered the active sites of thermostable B-family polymerases from thermophilic archaea to obtain new DNA polymerase mutants. By increasing the polymerase's incorporation efficiency for specific non-natural dNTPs, the inventors improved the sequencing speed and quality of sequencing by synthesis (SBS) methods.

[0009] To this end, in one aspect of the present application, a DNA polymerase mutant is provided. According to an embodiment of the present application, the mutant comprises: compared with a Pyrococcus sp. GB-D DNA polymerase exo-mutant, at least three amino acid mutations at the following four sites or functionally equivalent sites: position 409, position 410, position 411, and position 486; the Pyrococcus sp. GB-D DNA polymerase exo-mutant has an amino acid sequence as shown in SEQ ID NO: 1; the DNA polymerase mutant can effectively improve the polymerase's incorporation efficiency of specific non-natural dNTPs, thereby improving the sequencing speed and sequencing quality of the sequencing by synthesis (SBS) sequencing method.

[0010] In some examples, incorporation refers to the attachment of a modified nucleotide to the free 5' hydroxyl group of a second nucleotide via formation of a phosphodiester bond with the 3' phosphate group of the modified nucleotide. The second nucleotide to which the modified nucleotide is attached typically occurs at the 3' terminus of the polynucleotide chain. The terms "modified nucleotide" or "nucleotide analog" are equivalent and refer to a nucleotide that has been modified at the 3' sugar hydroxyl group such that the substituent is larger than the naturally occurring 3' hydroxyl group.

[0011] In some examples, the inventors found that mutations at one or two of positions 409, 410, 411, and 486 had little effect on enzyme activity (Table 1).

[0012] It should be noted that the amino acid positions in the amino acid sequence of the DNA polymerase mutant described in this application are located with reference to the amino acid positions in the amino acid sequence of the wild-type Pyrococcus sp. GB-D DNA polymerase or the wild-type Pyrococcus sp. GB-D DNA polymerase exo-mutant. The amino acid sequence shown in SEQ ID NO: 1 is obtained by mutating the amino acid D at position 141 of the wild-type Pyrococcus sp. GB-D DNA polymerase to A and the amino acid E at position 143 to A. The Pyrococcus sp. GB-D DNA polymerase exo-mutant obtained after these mutations does not have exonuclease activity, while the wild-type Pyrococcus sp. GB-D DNA polymerase has exonuclease activity.

[0013] It should be noted that the so-called functionally equivalent sites include sites where, under specific circumstances, amino acids or nucleotides mutate but the function or properties of the DNA polymerase do not change.

[0014] According to an embodiment of the present application, the above-mentioned DNA enzyme mutant may further include at least one of the following technical features:

[0015] According to an embodiment of the present application, the DNA polymerase mutant has at least 90% identity, or at least 91% identity, or at least 92% identity, or at least 93% identity, or at least 94% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity with the Pyrococcus sp. GB-D DNA polymerase exo-mutant, and in some preferred embodiments, has at least 100% identity; wherein, the DNA polymerase mutant does not include the following mutations simultaneously present: L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to I, and A at position 486 mutates to L.

[0016] According to an embodiment of the present application, the mutant has the following mutations:

[0017] (1) The amino acid L at position 409 is mutated to A, V, Y, I, F, Q, or M;

[0018] (2) amino acid Y at position 410 mutated to A or G;

[0019] (3) amino acid P at position 411 is not mutated or is mutated to Q, M, N, or C;

[0020] (4) The amino acid A at position 486 is mutated to N, P, T, E, L, F, K, H, R, Y, M, W, or I.

[0021] According to some specific embodiments of the present application, the sequencing recombinases (obtained from the DNA polymerase mutants described in the present application) produced by different mutation sites have different relative activities, which can provide more options for actual production needs.

[0022] According to an embodiment of the present application, the mutant has the following mutations: (1) amino acid L at position 409 is mutated to A or V or Y or I or F or Q or M; (2) amino acid Y at position 410 is mutated to A or G; (3) amino acid P at position 411 is not mutated or is mutated to Q or M or N or C; (4) amino acid A at position 486 is mutated to L or F or K or H or R or Y or M or W or I. In some examples of the present application, a combination of mutations having an enzyme activity greater than that of the control group is preferred.

[0023] According to an embodiment of the present application, the mutant has any one of the combined mutations (1)-(8):

[0024] (1) The amino acid Y at position 410 is mutated to G, the amino acid L at position 409 is mutated to V, the amino acid P at position 411 is not mutated, and the amino acid A at position 486 is mutated to L; or

[0025] (2) the amino acid Y at position 410 mutates to G, the amino acid L at position 409 mutates to A, the amino acid P at position 411 does not mutate, and the amino acid A at position 486 mutates to F; or

[0026] (3) amino acid Y at position 410 mutates to G, amino acid L at position 409 mutates to V, amino acid P at position 411 does not mutate, and amino acid A at position 486 mutates to F; or

[0027] (4) amino acid Y at position 410 mutates to G, amino acid L at position 409 mutates to Y, amino acid P at position 411 mutates to M, and amino acid A at position 486 mutates to K; or

[0028] (5) amino acid Y at position 410 is mutated to G, amino acid L at position 409 is mutated to V, amino acid P at position 411 is not mutated, and amino acid A at position 486 is mutated to Y; or

[0029] (6) amino acid Y at position 410 mutates to A, amino acid L at position 409 mutates to A, amino acid P at position 411 mutates to Q, and amino acid A at position 486 mutates to K; or

[0030] (7) amino acid Y at position 410 is mutated to A, amino acid L at position 409 is mutated to A, amino acid P at position 411 is not mutated, and amino acid A at position 486 is mutated to L; or

[0031] (8) The amino acid Y at position 410 mutated to A, the amino acid L at position 409 mutated to A, the amino acid P at position 411 did not mutate, and the amino acid A at position 486 mutated to W.

[0032] According to an embodiment of the present application, the mutant has any one of the combined mutations (1)-(11):

[0033] (1) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or

[0034] (2) L at position 409 is mutated to I, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or

[0035] (3) L at position 409 is mutated to A, Y at position 410 is mutated to A, P at position 411 is not mutated, and A at position 486 is mutated to H; or

[0036] (4) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to N, and A at position 486 mutates to K; or

[0037] (5) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to M, and A at position 486 mutates to L; or

[0038] (6) L at position 409 mutates to M, Y at position 410 mutates to G, P at position 411 does not mutate, and A at position 486 mutates to K; or

[0039] (7) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to Q, and A at position 486 mutates to F; or

[0040] (8) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to Q, and A at position 486 mutates to L; or

[0041] (9) L at position 409 mutates to A, Y at position 410 mutates to G, P at position 411 mutates to N, and A at position 486 mutates to K; or

[0042] (10) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to C, and A at position 486 mutates to L; or

[0043] (11) L at position 409 mutated to A, Y at position 410 mutated to A, P at position 411 mutated to N, and A at position 486 mutated to L.

[0044] According to an embodiment of the present application, the mutant has any one of the combined mutations (1)-(3):

[0045] (1) L at position 409 mutates to Y, Y at position 410 mutates to G, P at position 411 mutates to Q, and A at position 486 mutates to K; or

[0046] (2) L at position 409 is mutated to I, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to F; or

[0047] (3) L at position 409 mutated to A, Y at position 410 mutated to A, P at position 411 mutated to Q, and A at position 486 mutated to R.

[0048] According to specific embodiments of the present application, some mutants may have base bias in sequencing applications and can be adaptively selected based on actual experimental needs.

[0049] According to an embodiment of the present application, the mutant has any one of the combined mutations (1)-(10):

[0050] (1) L at position 409 is mutated to V, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or

[0051] (2) L at position 409 mutates to A, Y at position 410 mutates to G, P at position 411 does not mutate, and A at position 486 mutates to H; or

[0052] (3) L at position 409 mutates to F, Y at position 410 mutates to G, P at position 411 does not mutate, and A at position 486 mutates to F; or

[0053] (4) L at position 409 is mutated to V, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to H; or

[0054] (5) L at position 409 mutates to Q, Y at position 410 mutates to G, P at position 411 mutates to M, and A at position 486 mutates to K; or

[0055] (6) L at position 409 is mutated to Y, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to Y; or

[0056] (7) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to M; or

[0057] (8) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to I; or

[0058] (9) L at position 409 mutates to A, Y at position 410 mutates to G, P at position 411 mutates to M, and A at position 486 mutates to K; or

[0059] (10) L at position 409 mutated to A, Y at position 410 mutated to G, P at position 411 mutated to Q, and A at position 486 mutated to K.

[0060] According to some specific embodiments of the present application, the relative activity of the sequencing recombinase produced by the above-mentioned mutation sites is higher than that of the control group, resulting in faster speed and better quality in actual sequencing.

[0061] According to some specific embodiments of the present application, when the amino acid sequence of the DNA polymerase mutant has the above-mentioned mutation, the relative activity of the recombinant polymerase obtained compared to the existing mutation is stronger, and it can effectively add non-natural dNTP containing a fluorescently labeled 3'O-reversible terminator to the DNA chain, thereby improving the sequencing speed and sequencing quality.

[0062] It should be noted that the amino acid sequences of the DNA polymerase mutants referred to in any example of the present application are obtained by superimposing mutations on the sequence of SEQ ID NO: 1.

[0063] In a second aspect of the present application, a nucleic acid molecule is provided. According to an embodiment of the present application, the nucleic acid molecule encodes the DNA polymerase mutant described in the first aspect of the present application. The DNA polymerase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.

[0064] In the third aspect of the present application, the present application provides an expression vector. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule described in the second aspect of the present application.

[0065] It should be noted that the expression vector may further include a promoter, and the promoter is operably connected to the nucleic acid molecule.

[0066] According to an embodiment of the present application, the expression vector is a non-pathogenic viral vector, and the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.

[0067] In a fourth aspect of the present application, a recombinant cell is provided. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule described in the second aspect of the present application and the expression vector described in the third aspect of the present application. The recombinant cell is used to express or secrete the DNA polymerase mutant described in the first aspect of the present application.

[0068] According to an embodiment of the present application, the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

[0069] In a fifth aspect of the present application, a recombinant strain is provided. According to an embodiment of the present application, the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the present application. By culturing the recombinant strain, the DNA polymerase mutant can be rapidly obtained in large quantities.

[0070] In a sixth aspect of the present application, a method for obtaining a DNA polymerase mutant is provided. According to an embodiment of the present application, the method comprises culturing the recombinant cell described in the fourth aspect of the present application or the recombinant strain described in the fifth aspect of the present application under conditions suitable for protein expression, so as to obtain the DNA polymerase mutant.

[0071] In a seventh aspect of the present application, a complex is provided. According to an embodiment of the present application, the complex comprises the DNA polymerase mutant described in the first aspect of the present application and a small molecule compound or a macromolecule, wherein the DNA polymerase mutant and the small molecule compound or the macromolecule are coupled via a chemical bond.

[0072] According to an embodiment of the present application, the small molecule compound or macromolecule includes a fluorescent marker, fluorescein or an antibody, etc.

[0073] In an eighth aspect, the present application provides a method for nucleic acid synthesis. According to embodiments of the present application, the method comprises: subjecting a mixture of a nucleic acid template, an amplification primer, dNTPs, and the DNA polymerase mutant described in the first aspect of the present application to an amplification treatment under conditions suitable for nucleic acid amplification, thereby obtaining the nucleic acid. The aforementioned nucleic acid synthesis method enables efficient and rapid amplification of the nucleic acid template.

[0074] It should be noted that the DNA polymerase mutant described in the present application has polymerization activity for all dNTPs (including dNTPs with or without fluorescent labels).

[0075] In a ninth aspect of the present application, a method for nucleic acid sequencing is provided. According to an embodiment of the present application, the method comprises: subjecting a nucleic acid template to be tested to a mixed product of the DNA polymerase mutant and modified dNTPs described in the first aspect of the present application, performing an amplification process and a fluorescence signal detection process under conditions suitable for nucleic acid amplification; and determining the nucleic acid sequence of the nucleic acid to be tested based on the fluorescence signal obtained by detection.

[0076] In some examples of the present application, the modified dNTP is selected from non-natural dNTPs with a fluorescently labeled 3'O-reversible terminator.

[0077] Exemplarily, the nucleic acid sequencing method includes mixing a nucleic acid template to be tested with the DNA polymerase mutant and a non-natural dNTP with a fluorescently labeled 3'O-reversible terminator. The DNA polymerase mutant is responsible for matching the non-natural dNTP with the fluorescently labeled 3'O-reversible terminator with the nucleic acid template. Finally, by detecting multiple fluorescent labeling signals, a nucleic acid sequence obtained based on the obtained fluorescent labeling signals is obtained to obtain the nucleic acid sequence of the nucleic acid to be tested. The above polymerization reaction and fluorescent labeling reaction can be performed for multiple cycles depending on the length of the sequencing template.

[0078] In a tenth aspect of the present application, a nucleic acid sequencing kit is provided. According to embodiments of the present application, the nucleic acid sequencing kit includes the DNA polymerase mutant described in the first aspect or the complex described in the seventh aspect. The kit described in the present application is used for efficient, accurate, and rapid nucleic acid sequencing.

[0079] In an eleventh aspect of the present application, the present application provides a use of the nucleic acid sequencing kit of the ninth aspect in sequencing. According to embodiments of the present application, the kit can be used for sequencing, including but not limited to sequencing by synthesis (SBS) or sequencing by binding (SBB).

[0080] In the twelfth aspect of the present application, the present application proposes a use of the DNA polymerase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the recombinant strain described in the fifth aspect, or the complex described in the seventh aspect in the preparation of a product for catalyzing DNA amplification or nucleic acid sequencing. According to embodiments of the present application, the DNA polymerase mutant, nucleic acid molecule, expression vector, recombinant cell, recombinant strain, or complex can be used alone or in combination to prepare a product for catalyzing DNA amplification or nucleic acid sequencing.

[0081] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0083] FIG1 is a schematic diagram of the SDS-PAGE electrophoresis results of the Pyrococcus sp. GB-D exo-type DNA polymerase fusion protein described in one embodiment of the present application. DETAILED DESCRIPTION

[0084] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0085] In this application, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly indicate the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specified.

[0086] In this application, unless otherwise indicated, the term "amino acid" is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0087] Throughout this application, unless otherwise indicated, the term "nucleotide" encompasses both nucleotides and nucleosides. Nucleosides, like nucleotides, comprise a purine or pyrimidine base linked to a ribose or deoxyribose glycoside, but lack a phosphate residue. Synthetic and / or naturally occurring nucleotides, prior to modification of the 3' sugar hydroxyl group, are included within the definition.

[0088] In this application, unless otherwise indicated, the term "homology" has the conventional meaning in the art and refers to the "identity" between two nucleic acid or amino acid sequences, where the percentage represents the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences being compared after best alignment, with the differences between the two sequences being randomly distributed over their entire lengths. In this application, the mutants are described according to their mutations at specific residues, the positions of which are referenced to the positions of the amino acids in the amino acid sequence of wild-type Pyrococcus sp. GB-D DNA polymerase or a wild-type Pyrococcus sp. GB-D DNA polymerase exo-mutant.

[0089] In this application, unless otherwise specified, the term "relative activity" refers to the enzyme activity of each experimental group relative to the control group, calculated with the enzyme activity of the control group as 100%. The control group is a mutant of Pyrococcus sp. GB-D DNA polymerase exo-mutant (SEQ ID NO: 1) with the following amino acid mutations: L409A-Y410A-P411I-A486L; the experimental groups are mutants of Pyrococcus sp. GB-D DNA polymerase mutant (SEQ ID NO: 1) described in the Examples of this application with amino acid mutations at specific positions.

[0090] In this application, unless otherwise indicated, the term "transformation" refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of genes caused by the introduction of exogenous DNA into cells.

[0091] Studies have reported that DNA polymerases can be divided into three families, designated A, B, and C. Although the nucleotide binding sites of A and B family polymerases are structurally similar, their motifs differ significantly, reflecting their distinct recognition mechanisms for nucleotides and their analogs. Of particular note, thermostable B family polymerases from thermophilic archaea exhibit excellent incorporation performance for a variety of nucleotide analogs. These thermostable B family polymerases include KOD (Thermococcus kodakaraensis), 9°N (Thermococcus sp. 9°N), TGO (Thermococcus gorgonarius), TOK (Desulfurococcus sp. Tok), Vent DNA polymerase (Thermococcus litoralis), JDF-3, and pfu DNA polymerase (Pyrococcus furiosis).

[0092] To improve the polymerization speed and sequencing quality of existing DNA polymerases in high-throughput sequencing processes such as sequencing by synthesis (SBS), the inventors have discovered that while the primary amino acid sequence of the DNA polymerase active site of these enzymes remains conserved across different families, their incorporation efficiency for specific non-natural dNTPs (e.g., non-natural dNTPs containing 3'O-reversible terminators) can be enhanced through enzyme engineering. This approach is expected to improve the enzyme's ability to recognize and polymerize non-natural specific dNTPs while maintaining DNA polymerase activity.

[0093] By analyzing the protein structure and performing computational simulations on the wild-type polymerase sequence, the inventors identified regions in the Pyrococcus sp. GB-D DNA polymerase that are associated with non-natural dNTPs with fluorescent markers and 3'O-reversible terminators. The inventors then conducted targeted, rational design. This included: firstly, mutations at sites L409, Y410, and P411 to eliminate the steric gate effect at these sites, thereby improving the incorporation efficiency of non-natural dNTPs with sterically hindered 3'O-reversible terminators (such as methylazido); secondly, mutations at site A486 in the finger region to stabilize the tertiary ajar conformation of the non-natural dNTP with the polymerase, DNA template, and DNA primer, thereby reducing the activation energy required for the polymerization reaction and making it easier for the non-natural dNTP with a fluorescent group and reversible terminator to polymerize with DNA within the enzyme.

[0094] It is known that in other B family polymerases (eg, 9°N, Pfu, Vent, etc.), the mutant site combination with better 3'O-methylazido-dNTP polymerization activity is L409A-Y410A-P411I-A486L (patent US8460910B2).

[0095] This application uses the mutant site combination with better 3'O-methylazido-dNTP polymerization activity, L409A-Y410A-P411I-A486L, as the control group for further screening and optimization, aiming to find DNA polymerases with better performance so that they can more effectively interact with non-natural dNTPs with fluorescent labels and 3'O-reversible terminators, providing better conditions and efficiency for subsequent experiments.

[0096] In view of this, in one aspect, the present application provides a DNA polymerase mutant. Compared to a Pyrococcus sp. GB-D DNA polymerase exo-mutant, this mutant has at least three amino acid mutations at the following four sites or functionally equivalent sites: 409, 410, 411, and 486; the Pyrococcus sp. GB-D DNA polymerase exo-mutant has the amino acid sequence set forth in SEQ ID NO:1; wherein the DNA polymerase mutant does not include an L to A mutation at position 409, a Y to A mutation at position 410, a P to I mutation at position 411, or an A to L mutation at position 486. This DNA polymerase mutant exhibits significant activity and thermal stability advantages, effectively enhancing the ability to polymerize non-natural dNTPs with 3' O-reversible terminators, thereby significantly improving sequencing speed and quality.

[0097] In another aspect of the present application, the present application provides a nucleic acid molecule encoding the aforementioned DNA polymerase mutant.

[0098] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0099] In another aspect of the present application, an expression vector is provided, comprising the aforementioned nucleic acid molecule. The type of expression vector is not particularly limited, as long as it can replicate and express the corresponding mutant in a host cell.

[0100] In another aspect, the present application provides a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector, or expressing a DNA polymerase mutant. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently express the aforementioned DNA polymerase mutant under appropriate conditions.

[0101] In another aspect of the present application, a recombinant strain expressing the aforementioned DNA polymerase mutant is provided, wherein the recombinant strain can be propagated in large quantities in a short period of time and efficiently prepare the DNA polymerase mutant.

[0102] In another aspect of the present application, the present application proposes a method for obtaining a DNA polymerase mutant, wherein the aforementioned recombinant cell is cultured under conditions suitable for protein expression to obtain the DNA polymerase mutant.

[0103] In yet another aspect of the present application, a complex is provided, comprising the aforementioned DNA polymerase mutant and a small molecule compound or a macromolecule, wherein the DNA polymerase mutant and the small molecule compound or macromolecule are chemically coupled. In the field of gene sequencing, the nucleic acid sequence to be detected is determined by detecting the small molecule compound (fluorescent marker) or macromolecule (antibody, fluorescein) in the complex.

[0104] It should be noted that the application scenarios of the DNA polymerase mutants and complexes proposed in this application include but are not limited to nucleic acid synthesis and nucleic acid sequencing. They can also be used to screen drugs targeting viruses or cell division, and have great development prospects in forensic medicine and criminology.

[0105] The present invention will be described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not indicate the manufacturer are conventional products that can be obtained commercially.

[0106] Example 1

[0107] This example is based on the preparation and crude enzyme activity screening of Pyrococcus sp. GB-D exo-type (Pyrococcus GB-D exo-mutant) DNA polymerase and its mutants. The specific steps are as follows:

[0108] 1. Construction and transformation of recombinant expression vector

[0109] To construct a recombinant expression vector, the pD441-WT vector plasmid containing the Pyrococcus sp. GB-D exo-type DNA polymerase gene (SEQ ID NO: 2) fused with a C-terminal 6x His tag and site-directed mutagenesis primers at sites 409, 410, 411, and 486 were synthesized at Sangon Biotech (Shanghai) Co., Ltd.

[0110] The DNA sequence of the wild-type Pyrococcus sp. GB-D DNA polymerase exo- mutant (WT-Exo-) is shown in SEQ ID NO: 2:

[0111] The primer design principle is: 5'-reverse complementary region (15-21 bp)-non-complementary region (at least 15 bp). Phanta Max Super-Fidelity DNA Polymerase was used to amplify the target plasmid.

[0112] Among them, positions 409, 410, 411, and 486 were mutated to the following amino acids:

[0113] The amino acid at position 409 is mutated to any amino acid;

[0114] amino acid 410 mutated to A or G;

[0115] Amino acid 411 is wild-type P, or mutated to one of C, E, M, N, or Q;

[0116] Amino acid 486 mutated to any amino acid

[0117] One known mutation combination that exhibits optimal polymerization activity for 3'-reversible terminators of non-natural dNTPs in B-family polymerases (e.g., 9°N, Pfu, and Vent) is L409A-Y410A-P411I-A486L (patents EP1664287B1 and US8460910B2). In this example, this mutation combination is designated Mut-2-1 (control group) and used as a control mutation site combination in a Pyrococcus sp. GB-D exo-type DNA polymerase screening experiment.

[0118] Because the amplified product contains the original template plasmid, in order to prevent the formation of false positive transformants after transformation, Dpn I digestion must be performed before recombination circularization to remove the methylated template plasmid.

[0119] The recombinant polymerase mutant expression vector was introduced into Escherichia coli BL21 competent cells. The cells were smeared on a resistance plate (containing 50 μg / ml of kanamycin) to screen for positive colonies. After overnight culture, single colonies of the experimental group were selected and cultured overnight in 5 ml of LB medium containing 50 μg / ml of kanamycin at 37°C and 200 rpm / min. The next day, the bacteria were cultured in a 96-well cell plate containing 150 μl of fresh LB medium and 50 μg / ml of kanamycin at a dilution ratio of 1:100 and shaken at 37°C and 220 rpm until OD600 = 0.6.

[0120] 2. Recombinant protein induction expression and bacterial lysis

[0121] Induce expression by adding IPTG to a final concentration of 0.5 mM in the above well plate at 25°C, 220 rpm / min, and incubate for 12-16 hours. Then, add 1 μL of 100 mM PMSF (a protease inhibitor) and 5 μL of 10 mg / ml lysozyme. Gently pipette and mix thoroughly. Incubate at 37°C for 10 minutes to obtain a crude enzyme solution.

[0122] 3. Fret recombinant protein-non-natural dNTP incorporation efficiency test

[0123] The crude enzyme solution was added with AF532 fluorescent dye-labeled 3'O-azidomethyl blocked dATP (MGI patent US10988501B2) and Cy5 fluorescent dye-labeled DNA chain (Shanghai Bioengineering). The sequence information is as follows:

[0124] F: Cy5-CGTGTATGCGTAATAGGATCCCGACTCACTATGGACG;

[0125] R: Cy5-CGTGTATCGTCCATAGTGAGTCGGGATCCTATTACGC;

[0126] The incorporation of modified nucleotides during high-throughput sequencing was simulated, and the relative reaction rates of polymerase mutants were detected using a microplate reader to calculate crude enzyme activity. The specific experimental method is as follows: Reaction Buffer: 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, pH 8.5@25℃.

[0127] The reaction system is as follows: 1 μg of different polymerase proteins obtained in step 2 are mixed with reaction solutions containing 2 μM modified dATP and 1 μM DNA-Cy5.

[0128] The reaction solution formula is: 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 2μM 3'-blocked modified dATP and 1μM DNA-Cy5, the balance is water, pH 8.5.

[0129] Reaction temperature: 60°C

[0130] Reaction time: 1h.

[0131] After the reaction is complete, data tables and enzyme activity curves can be directly exported to calculate the relative activity (Relative Vmax / Km) of the crude enzyme compared to the control enzyme mutant (Mut-2-1). The results, shown in Table 1, show that wild-type Pyrococcus sp. GB-D DNA polymerase (WT-Exo-) cannot incorporate 3'-O-azidomethyl-blocked dATP labeled with AF532 fluorescent dye, while some mutant proteins can incorporate it, generating fluorescent signals. The experimental results show that different mutants have different polymerization activities toward modified 3'-blocked dNTPs, indicating effective mutations and site combinations at different sites. Mutation site combinations with higher Ralative Vmax / Km values ​​than the control site are considered mutants that efficiently incorporate 3'-blocked dNTPs. These mutants offer great potential for high-throughput sequencing. Table 1 lists some of the mutation combinations at four sites in the DNA polymerase mutants. The control mutant, Mut-2-1, includes the combination L409A-Y410A-P411I-A486L.

[0132] Table 1: DNA polymerase mutant combination types

[0133] Example 2

[0134] According to the examples of the present application, the expression and purification of the fusion protein based on the Pyrococcus sp. GB-D exo-type DNA polymerase mutant were performed. The specific steps are as follows:

[0135] 1) Pick a single BL21 / pD441 mutant colony and culture it in 50 ml of LB liquid culture medium (containing 50 μg / ml kanamycin) at 37°C, 220 rpm / min, overnight. The next day, dilute the culture 1:100 and transfer it to 1000 ml of LB liquid culture medium (containing 50 μg / ml kanamycin). Cultivate the culture at 37°C, 220 rpm / min, and shake until the OD600 reaches 0.5-0.8. Add IPTG to a final concentration of 0.5 mM and induce the culture overnight at 25°C. Collect the induced BL21 / pD441 culture. Simultaneously, collect the uninduced BL21 / pD441 culture without IPTG as a blank control.

[0136] 2) The BL21 / pD441 bacterial suspension induced in step 1) was centrifuged at 8000 rpm / min for 10 min, the supernatant was discarded, the precipitated cells were collected, and the cells were resuspended in buffer 1 (50 mM KPO4, 500 mM NaCl, 10 mM imidazole, 5% Glycerol, pH 7.0), and PMSF (final concentration 0.5 mM) and Triton X-ray distillation were added. X-100 (final concentration 0.5%) and Lysozyme (lysozyme, final concentration 0.25%) were added, followed by incubation at room temperature for 30 min, centrifugation at 12,000 rpm / min and 4°C for 30 min, ice bath, and ultrasonic cell disruption; centrifugation at 12,000 rpm / min for 30 min, and the supernatant obtained by centrifugation was placed in a 75°C water bath for 20 min, with regular mixing during the period to ensure uniform heating; centrifugation at 12,000 rpm / min and 4°C for 30 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain a crude fusion protein extract.

[0137] 3) The crude fusion protein extract obtained in step 2) was loaded onto a Ni column affinity chromatography (affinity chromatography prepacked column HisTrap FF, 5 ml, 17-5255-01, GE healthcare) at an appropriate flow rate. After loading, the column was equilibrated with buffer 1 for 5 CV (column volume); eluted with 3% buffer 2 (50 mM KPO4, 1 M NaCl, 5% Glycerol, pH 7.0) for 5 CV; and eluted with 50% buffer 2 for 5 CV. The Ni column affinity chromatography eluate corresponding to the peak value greater than or equal to 100 mAU was collected.

[0138] 4) The eluate corresponding to the peak with a concentration of 100 mAU or greater was loaded at a specific flow rate for ion exchange chromatography (prepacked ion exchange column HiTrap Q HP, 5 ml, 17-1154-01, GE healthcare). After loading, the column was equilibrated with buffer 2 for 5 CV and linearly eluted from 0% buffer 2 to 60% buffer 2. The eluate corresponding to the peak with a concentration of 100 mAU or greater was collected.

[0139] 5) The ion exchange chromatography eluate corresponding to the peak of 100 mAU or more was subjected to gel chromatography (gel chromatography prepacked column HiPrep Sephacryl S-100HR, 26 mm, 17-1194-01, GE healthcare). The column was first washed with 20% ethanol for 3 CV, then washed with water for 3 CV, equilibrated with 100% buffer 3 (20 mM Tris, 200 mM KCl, 0.2 mM EDTA, 10% Glycerol, pH 7.4) for 3 CV, and then loaded. The column was then eluted with buffer 3 for 1.5 CV, and the eluate was collected to obtain the purified wild-type DNA polymerase fusion protein.

[0140] 6) The Pyrococcus sp. GB-D exo-type DNA polymerase fusion protein was subjected to SDS-PAGE (5% stacking gel, 12% resolving gel). The results are shown in Figure 1. Lane 1 is a protein marker (PageRuler Prestained Protein Ladder, 26616, Thermo Scientific), lane 2 is 5 μl of lysate from induced BL21 / pD441 bacterial culture carrying the Pyrococcus sp. GB-D exo-type DNA polymerase mutant fusion protein plasmid mixed with 10 μl of 2x loading buffer, lane 3 is 5 μl of flow-through from the lysate after Ni column affinity chromatography mixed with 10 μl of 2x loading buffer, and lane 4 is 0.5 μl of the Pyrococcus sp. GB-D exo-type DNA polymerase mutant fusion protein purified by Ni column affinity chromatography mixed with 1 μl of 2x loading buffer. As can be seen, the protein in lanes 2, 3, and 4 is approximately 90 kDa in size, consistent with the molecular weight reported in the literature (Hikida Y, Kimoto M, Hirao I, Yokoyama S. Crystal structure of Deep Vent DNA polymerase. Biochemical and biophysical research communications. 2017 Jan 29; 483(1): 52-7). Protein purity was analyzed using Quantity One software on the gel after electrophoresis. The purity of the purified DNA polymerase mutant fusion protein reached 90% or higher. The target protein of approximately 90 kDa was not obtained in the uninduced BL21 / pD441 bacterial culture.

[0141] Example 3

[0142] According to the examples of this application, the actual sequencing performance of pure enzymes of Pyrococcus sp. GB-D exo-type DNA polymerase mutants was tested on a sequencer. The purified enzymes of the dominant mutants were sequenced on a BGI MGISEQ-2000 sequencer, and the sequencing results were analyzed. The specific experimental steps are as follows:

[0143] Sequencing library: Ecoli.fa library (Part No. 1000005038, MGI Tech co., Ltd);

[0144] Sequencing conditions: polymerization time 60 seconds, excision time 60 seconds.

[0145] Using the same sequencing library, Ecoli.fa, and sequencing conditions, we selected a control Mut1 mutant and five mutants that exhibited varying performance in the enzyme activity test for on-line testing. These mutants were sequenced for 100 cycles (SE100) on the MGISEQ-2000 sequencer. The test results, shown in Table 2, demonstrate that the purified enzyme from the dominant mutant performed better in actual sequencing.

[0146] Table 2:

[0147] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0148] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A DNA polymerase mutant, characterized in that: Compared with a Pyrococcus sp. GB-D DNA polymerase exo-mutant, it has at least three amino acid mutations in the following four positions or functionally equivalent positions: position 409, position 410, position 411, and position 486; The Pyrococcus sp. GB-D DNA polymerase exo-mutant has an amino acid sequence as shown in SEQ ID NO:

1.

2. The DNA polymerase mutant according to claim 1, characterized in that The DNA polymerase mutant has at least 95% identity, at least 98% identity, or at least 99% identity, preferably at least 100% identity with the Pyrococcus sp. GB-D DNA polymerase exo-mutant, except for the amino acid sequences at positions 409, 410, 411, and 486; wherein the DNA polymerase mutant does not include the following mutations co-existing: The 409th position L mutated to A, the 410th position Y mutated to A, the 411th position P mutated to I, and the 486th position A mutated to L.

3. The DNA polymerase mutant according to claim 1, characterized in that The mutant has the following mutations: (1) The amino acid L at position 409 is mutated to A, V, Y, I, F, Q or M; (2) amino acid Y at position 410 mutated to A or G; (3) the amino acid P at position 411 does not mutate or mutates to Q, M, N, or C; (4) The amino acid A at position 486 is mutated to N, P, T, E, L, F, K, H, R, Y, M, W, or I.

4. The DNA polymerase mutant according to claim 3, characterized in that The mutant has the following mutations: (1) The amino acid L at position 409 is mutated to A, V, Y, I, F, Q or M; (2) amino acid Y at position 410 mutated to A or G; (3) the amino acid P at position 411 does not mutate or mutates to Q, M, N, or C; (4) The amino acid A at position 486 is mutated to L, F, K, H, R, Y, M, W or I.

5. The DNA polymerase mutant according to claim 4, characterized in that The mutant has any one of the following combined mutations (1)-(8): (1) the amino acid Y at position 410 is mutated to G, the amino acid L at position 409 is mutated to V, the amino acid P at position 411 is not mutated, and the amino acid A at position 486 is mutated to L; or (2) the amino acid Y at position 410 is mutated to G, the amino acid L at position 409 is mutated to A, the amino acid P at position 411 is not mutated, and the amino acid A at position 486 is mutated to F; or (3) the amino acid Y at position 410 is mutated to G, the amino acid L at position 409 is mutated to V, the amino acid P at position 411 is not mutated, and the amino acid A at position 486 is mutated to F; or (4) the amino acid Y at position 410 mutates to G, the amino acid L at position 409 mutates to Y, the amino acid P at position 411 mutates to M, and the amino acid A at position 486 mutates to K; or (5) the amino acid Y at position 410 is mutated to G, the amino acid L at position 409 is mutated to V, the amino acid P at position 411 is not mutated, and the amino acid A at position 486 is mutated to Y; or (6) the amino acid Y at position 410 mutates to A, the amino acid L at position 409 mutates to A, the amino acid P at position 411 mutates to Q, and the amino acid A at position 486 mutates to K; or (7) the amino acid Y at position 410 is mutated to A, the amino acid L at position 409 is mutated to A, the amino acid P at position 411 is not mutated, and the amino acid A at position 486 is mutated to L; or (8) The amino acid Y at position 410 mutated to A, the amino acid L at position 409 mutated to A, the amino acid P at position 411 did not mutate, and the amino acid A at position 486 mutated to W.

6. The DNA polymerase mutant according to claim 4, characterized in that The mutant has any one of the combined mutations (1) to (11): (1) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or (2) L at position 409 is mutated to I, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or (3) L at position 409 is mutated to A, Y at position 410 is mutated to A, P at position 411 is not mutated, and A at position 486 is mutated to H; or (4) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to N, and A at position 486 mutates to K; or (5) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to M, and A at position 486 mutates to L; or (6) L at position 409 is mutated to M, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or (7) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to Q, and A at position 486 mutates to F; or (8) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to Q, and A at position 486 mutates to L; or (9) L at position 409 mutates to A, Y at position 410 mutates to G, P at position 411 mutates to N, and A at position 486 mutates to K; or (10) L at position 409 mutates to A, Y at position 410 mutates to A, P at position 411 mutates to C, and A at position 486 mutates to L; or (11) L at position 409 mutated to A, Y at position 410 mutated to A, P at position 411 mutated to N, and A at position 486 mutated to L.

7. The DNA polymerase mutant according to claim 4, characterized in that The mutant has any one of the combined mutations (1) to (3): (1) L at position 409 mutates to Y, Y at position 410 mutates to G, P at position 411 mutates to Q, and A at position 486 mutates to K; or (2) L at position 409 is mutated to I, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to F; or (3) L at position 409 mutated to A, Y at position 410 mutated to A, P at position 411 mutated to Q, and A at position 486 mutated to R.

8. The DNA polymerase mutant according to claim 3, characterized in that The mutant has any one of the combined mutations (1) to (10): (1) L at position 409 is mutated to V, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to K; or (2) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to H; or (3) L at position 409 mutates to F, Y at position 410 mutates to G, P at position 411 does not mutate, and A at position 486 mutates to F; or (4) L at position 409 is mutated to V, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to H; or (5) L at position 409 mutates to Q, Y at position 410 mutates to G, P at position 411 mutates to M, and A at position 486 mutates to K; or (6) L at position 409 is mutated to Y, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to Y; or (7) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to M; or (8) L at position 409 is mutated to A, Y at position 410 is mutated to G, P at position 411 is not mutated, and A at position 486 is mutated to I; or (9) L at position 409 mutates to A, Y at position 410 mutates to G, P at position 411 mutates to M, and A at position 486 mutates to K; or (10) L at position 409 mutated to A, Y at position 410 mutated to G, P at position 411 mutated to Q, and A at position 486 mutated to K.

9. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the DNA polymerase mutant according to any one of claims 1 to 8.

10. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 9.

11. The expression vector according to claim 10, characterized in that The expression vector is a non-pathogenic viral vector; The non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.

12. A recombinant cell, characterized in that Carrying the nucleic acid molecule according to claim 10 or the expression vector according to claim 11.

13. The recombinant cell according to claim 12, characterized in that The recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

14. A recombinant strain, characterized in that The recombinant strain expresses the DNA polymerase mutant according to any one of claims 1 to 8.

15. A method for obtaining a DNA polymerase mutant, characterized in that: include: The recombinant cell according to any one of claims 12 to 13 or the recombinant strain according to claim 14 is cultured under conditions suitable for protein expression to obtain the DNA polymerase mutant.

16. A composite, characterized in that The invention comprises the DNA polymerase mutant according to any one of claims 1 to 8 and a small molecule compound or a macromolecule, wherein the mutant and the small molecule compound or the macromolecule are coupled via a chemical bond.

17. The composite according to claim 16, characterized in that The small molecule compound or macromolecule includes a fluorescent marker, fluorescein or an antibody.

18. A method for synthesizing nucleic acid, characterized in that: include: The mixed product of nucleic acid template, amplification primer, dNTP and the DNA polymerase mutant according to any one of claims 1 to 8 is subjected to amplification treatment under conditions suitable for nucleic acid amplification to obtain the nucleic acid.

19. A method for nucleic acid sequencing, characterized in that: include: Amplify the nucleic acid to be tested and the mixed product of the DNA polymerase mutant according to any one of claims 1 to 8 and the modified dNTP under conditions suitable for nucleic acid amplification and perform fluorescence signal detection treatment; as well as Based on the fluorescent signal obtained by detection, the nucleic acid sequence of the nucleic acid to be detected is determined.

20. A nucleic acid sequencing kit, characterized in that: It comprises the DNA polymerase mutant according to any one of claims 1 to 8 or the complex according to claim 16 or 17.

21. Use of the nucleic acid sequencing kit according to claim 20 in sequencing.

22. Use of the DNA polymerase mutant according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to any one of claims 10 to 11, the recombinant cell according to any one of claims 12 to 13, the recombinant strain according to claim 14 or the complex according to any one of claims 16 to 17 in the preparation of products related to catalysis of DNA amplification or nucleic acid sequencing.

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