DNA polymerase mutant and use thereof

Abstract

The present invention provides a DNA polymerase mutant and a use thereof. The DNA polymerase mutant has DNA polymerization activity, and comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO: 1. The invention can solve the problem of low polymerization activity of wild-type DNA polymerases in the prior art, and is applicable in the field of DNA polymerases.

Description

DNA polymerase mutants and their applications Technical Field

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

[0002] PCR technology is a cornerstone of modern molecular biology, and Taq DNA polymerase is a classic workhorse of PCR. Taq DNA polymerase belongs to the A family of DNA polymerases and exhibits excellent thermostability, making it widely used in PCR-related fields. While lacking 3'-5' proofreading activity, Taq polymerase possesses 5'-3' exonuclease activity and the ability to add A residues to its terminal ends. It is widely used in molecular biology applications such as direct PCR, allele detection, Sanger sequencing, fluorescent quantitative PCR, and TA cloning. Currently, PCR is one of the fastest-growing technologies in the molecular biology application market. New applications and techniques for PCR are being developed and applied in research and diagnostics. With the widespread adoption of PCR, research on the properties of Taq DNA polymerase is becoming increasingly important. However, Taq DNA polymerase also has some limitations, such as its amplification activity and inhibitor resistance, which still require further improvement. There is a continued need for improved DNA polymerase variants that combine the necessary properties to meet the requirements of DNA amplification, synthesis, detection, sequencing, and other important technologies.

[0003] Summary of the Invention

[0004] The main purpose of the present invention is to provide a DNA polymerase mutant and its application to solve the problem of low polymerization activity of wild-type DNA polymerase in the prior art.

[0005] To achieve the above-mentioned object, according to a first aspect of the present invention, a DNA polymerase mutant is provided, which has DNA polymerase activity and comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology to the amino acid sequence shown in SEQ ID NO: 1.

[0006] Furthermore, the DNA polymerase mutant has an amino acid substitution at at least one of the following sites relative to the amino acid sequence shown in SEQ ID NO: 1: D488, S577, S543, E388, E400, E397, T544, L549, V586, L670, L678, T664 and G59.

[0007] Furthermore, the amino acid substitution at the D488 site includes D488H, D488R or D488K; the amino acid substitution at the S543 site includes S543T; the amino acid substitution at the S577 site includes S577T; the amino acid substitution at the E388 site includes E388K; the amino acid substitution at the E400 site includes E400R; the amino acid substitution at the E397 site includes E397Q; the amino acid substitution at the T544 site includes T544Y, T544D, T544K, T544S, ... 544W or T544F; amino acid substitution at L549 site includes L549F, L549I, L549D or L549K; amino acid substitution at V586 site includes V586F; amino acid substitution at L670 site includes L670F; amino acid substitution at L678 site includes L678Y, L678W, L678I, L678F, L678K or L678D; amino acid substitution at T664 site includes T664F; amino acid substitution at G59 site includes G59W.

[0008] Furthermore, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: D488H, D488R, D488K, S577T, G59W, E388K, E397Q, E400R, S543T, T544Y, T544D, T544K, T544S, T544W, T544F, L549F, L549I, L549D, L549K, V586F, T664F, L670F, L678Y, L678K, L678D, L678I, L678F, G59W+E388K, G59W+E400R, G59W+D488K, G59W+S543T, G59W+S577T, E388K+E397Q, E388K+E400R, E388K+D488K, E388K+S543T, E38 8K+T544Y, E388K+L549F, E388K+S577T, E388K+L670F, E388K+T664F, E388K+L678Y, E397Q+D488K, E397Q+T664F, E4 00R+D488K, E400R+T664F, D488K+S543T, D488K+T544Y, D488K+L549F, D488K+S577T, D488K+V586F, D488K+T664F, D 488K+L670F, D488K+L678Y, S543T+T664F, S577T+T664F, V586F+T664F, T664F+L670F, T664F+L678W, T664F+L678Y, G59W+D488K+S543T or G59W+D488K+L549F; preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W, D488K, G59W+D488K, S543T, E 388K, E400R, E397Q, L549F, T664F, G59W+D488K+L549F, G59W+S577T, G59W+E388K, G59W+E400R, E400R+D488K, D488K+L549F, E388K+E397Q, E388K+T664F, S543T+T664F, E400R+T664F, L678I or E388K+L549F, the DNA polymerase mutant has a DNA polymerization activity that is better than the DNA polymerization activity of the amino acid sequence set forth in SEQ ID NO: 1;Preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W+D488K, S543T, E400R, T544Y, L549F, G59W+D488K+L549F, G59W+S543T, G59W+S577T or G59W+E400R, and the multiple amplification ability of the DNA polymerase mutant is better than that of SEQ ID NO: 1; preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W, G59W+D488K, S543T, S577T, E400R, L549F, T664F, G59W+D488K+S543T, G59W+D488K+L549F, G59W+S577T, G59W+E388K, S543T+T664F, L678I, E388K+L549F, G59W+D488K, G59W+D488K+L549F, G59W+S577T, or G59W+E388K, and the DNA polymerase mutant has a better resistance to PCR inhibitors than the amino acid sequence shown in SEQ ID NO: 1.

[0009] In order to achieve the above object, according to a second aspect of the present invention, a DNA molecule is provided, which comprises a polynucleotide encoding the above DNA polymerase mutant.

[0010] In order to achieve the above object, according to the third aspect of the present invention, a recombinant vector is provided, which contains the above DNA molecule.

[0011] In order to achieve the above object, according to the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or the above recombinant vector.

[0012] Furthermore, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0013] In order to achieve the above object, according to a fifth aspect of the present invention, a PCR kit is provided, which comprises the above DNA polymerase mutant.

[0014] Furthermore, the PCR kit further includes any one or more of the following components: 1) a buffer for providing a PCR amplification environment; 2) PCR primers; 3) a reagent for extracting target DNA.

[0015] In order to achieve the above object, according to a sixth aspect of the present invention, a PCR method is provided, which comprises: using the above DNA polymerase mutant or the above PCR kit to perform PCR amplification on the target DNA.

[0016] Furthermore, PCR includes multiplex PCR amplification; preferably, the PCR system contains a PCR inhibitor; more preferably, the PCR inhibitor includes one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions and iron ions.

[0017] The activity of DNA polymerase is one of the key factors in improving PCR amplification efficiency. By applying the technical solution of the present invention, the DNA polymerase mutant has higher activity than the wild-type Taq DNA polymerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] FIG1 shows the agarose gel electrophoresis results according to Example 4 of the present invention.

[0020] FIG2 shows the agarose gel electrophoresis results according to Example 4 of the present invention.

[0021] FIG3 shows the agarose gel electrophoresis results according to Example 4 of the present invention.

[0022] FIG4 shows the agarose gel electrophoresis results according to Example 5 of the present invention. DETAILED DESCRIPTION

[0023] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] As mentioned in background technology, with the widespread application of PCR technology, the research on DNA polymerase properties is becoming increasingly important. However, there are also some shortcomings in DNA polymerase, and the polymerization activity is difficult to meet the needs of practical applications, and it is urgent to improve DNA polymerase, thereby improving DNA polymerization rate. Polymerization rate, or the number of nucleotides incorporated per unit time (under specified reaction conditions, including temperature, pH, ionic strength, etc.), is affected by many parameters, including the binding affinity (dNTP, primer) and catalytic efficiency (nucleotide transfer rate, pyrophosphate release and translocation step) of the substrate. In the present application, the inventor attempts to develop a novel DNA polymerase mutant with enhanced polymerization activity, thereby proposing a series of protection schemes of the present application.

[0025] In a first typical embodiment of the present application, a DNA polymerase mutant is provided, which has DNA polymerase activity and includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology to the amino acid sequence shown in SEQ ID NO: 1.

[0026] SEQ ID NO: 1:

[0027] In a preferred embodiment, the above-mentioned DNA polymerase mutant has an amino acid substitution at at least one of the following sites relative to the amino acid sequence shown in SEQ ID NO: 1: D488, S577, S543, E388, E400, E397, T544, L549, V586, L670, L678, T664 and G59.

[0028] In a preferred embodiment, the amino acid substitution at site D488 includes D488H, D488R or D488K; the amino acid substitution at site S543 includes S543T; the amino acid substitution at site S577 includes S577T; the amino acid substitution at site E388 includes E388K; the amino acid substitution at site E400 includes E400R; the amino acid substitution at site E397 includes E397Q; the amino acid substitution at site T544 includes T544Y, T544D, T544K, T544S, T544W or T544F; Amino acid substitutions at site 549 include L549F, L549I, L549D or L549K; amino acid substitutions at site V586 include V586F; amino acid substitutions at site L670 include L670F; amino acid substitutions at site L678 include L678Y, L678W, L678I, L678F, L678K or L678D; amino acid substitutions at site T664 include T664F; amino acid substitutions at site G59 include G59W; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0029] In a preferred embodiment, the mutation (substitution) of the DNA polymerase mutant includes any one of the following amino acid mutation combinations:

[0030] G59W, E388K, E397Q, E400R, D488H, D488R, D488K, S543T, T544Y, T544D, T544K, T544S, T5 44W, T544F, L549F, L549I, L549D, L549K, S577T, V586F, T664F, L670F, L678Y, L678K, L67 8D, L678I, L678F, G59W+E388K, G59W+E400R, G59W+D488K, G59W+S543T, G59W+S577T, E38 8K+E397Q, E388K+E400R, E388K+D488K, E388K+S543T, E388K+T544Y, E388K+L549F, E388K +S577T, E388K+L670F, E388K+T664F, E388K+L678Y, E397Q+D488K, E397Q+T664F, E400R+ D488K, E400R+T664F, D488K+S543T, D488K+T544Y, D488K+L549F, D488K+S577T, D488K+V 586F, D488K+T664F, D488K+L670F, D488K+L678Y, S543T+T664F, S577T+T664F, V586F+T664F, T664F+L670F, T664F+L678W, T664F+L678Y, G59W+D488K+S543T, or G59W+D488K+L549F;

[0031] Preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W, D488K, G59W+D488K, S543T, E388K, E400R, E397Q, L549F, T664F, G59W+D488K+L549F, G59W+S577T, G59W+E388K, G59W+E400R, E400R+D488K, D488K+L549F, E388K+E397Q, E388K+T664F, S543T+T664F, E400R+T664F, L678I or E388K+L549F.

[0032] The enhanced polymerase activity of the DNA polymerase mutant can produce more double-stranded DNA products per unit time compared to the wild-type DNA polymerase (SEQ ID NO: 1).

[0033] Preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W+D488K, S543T, E400R, T544Y, L549F, G59W+D488K+L549F, G59W+S543T, G59W+S577T or G59W+E400R.

[0034] Multiplex PCR refers to a technique that simultaneously amplifies multiple targets through a single PCR reaction and then detects the amplified products using a specific detection method, thereby enabling diagnosis of multiple targets. The advantage of multiplex PCR technology lies in its high efficiency. A single amplification reaction can detect and identify multiple pathogens, significantly reducing the cost compared to multiple single-plex assays. It is widely used in scientific research and disease diagnosis, and is the most widely used multiplex nucleic acid detection technology in clinical practice. It is currently primarily used for the detection and quantification of bacteria and viruses, single nucleotide polymorphism (SNP) typing, disease mutation gene detection, and promoter methylation. Since Chamberlain first proposed this concept in 1988, multiplex PCR technology has developed rapidly. ThermoFisher's multiplex PCR kit can achieve 20-plex amplification; software such as PrimerPlex and iCubate2.0 can facilitate the design and optimization of multiplex PCR primers and probes, thereby reducing the possibility of nonspecific amplification. Liao et al. successfully used fluorescent probe melting curve technology to type 15 high-risk HPV types and type and analyze 48 human SNPs.

[0035] The basic principle of multiplex PCR is the same as that of conventional PCR. The difference is that two or more pairs of primers are added to the multiplex PCR reaction system, and each pair of primers binds to the corresponding part of the template respectively, and simultaneously amplifies the PCR reaction of multiple nucleic acid fragments. However, the multiplex PCR experiment is not simply mixing multiple pairs of specific primers into a reaction system. The difficulty lies in the balanced amplification of each target in the system. In multiplex PCR, the amplification conditions may be incompatible between multiple targets, and non-specific amplification may exist under multiple primers. In order to achieve balanced amplification of each target, primers, reaction conditions, reaction systems, etc. are generally optimized. As the core raw material in multiplex PCR applications, DNA polymerase, in addition to conventional PCR performance, also needs to have amplification uniformity and equal preference for various primers. Otherwise, some products will be very large in amount and can be easily detected, while some products will be completely reduced to background.

[0036] Compared with the wild-type DNA polymerase (SEQ ID NO: 1), the above-mentioned DNA polymerase mutant has better multiplex amplification ability, that is, it meets the amplification rate requirements in multiplex PCR and has amplification uniformity. It can perform PCR amplification with the same preference for different primers and target fragments, thereby achieving the effect of simultaneously amplifying multiple targets in a single PCR reaction.

[0037] Preferably, the mutation includes any one of the following amino acid mutation combinations: G59W, G59W+D488K, S543T, S577T, E400R, L549F, T664F, G59W+D488K+S543T, G59W+D488K+L549F, G59W+S577T, G59W+E388K, S543T+T664F, L678I, E388K+L549F, G59W+D488K, G59W+D488K+L549F, G59W+S577T or G59W+E388K.

[0038] PCR, one of the most important tools in molecular biology research, amplifies not only purified DNA templates but also a wide variety of complex templates. Most wild-type polymerases are restricted by PCR inhibitors in the amplification template, affecting their amplification capacity. These inhibitors include humic acid in soil, various components in serum, and animal and plant tissues. This limitation hinders the more efficient and widespread application of PCR in various fields.

[0039] PCR-based methods and techniques, such as those used in clinical diagnostics, environmental testing, and forensic analysis, utilize a variety of complex sample types, including blood, blood cards, oral mucosal cells (buccal swabs), soil, and plant tissue. These samples often contain inhibitory substances that can interfere with PCR, leading to false-negative reactions or reduced sensitivity. PCR analysis of blood samples is commonly used in clinical settings, including for diagnosing genetic diseases, viral and microbial infections, blood typing, and human health testing for blood banking. To minimize the impact of inhibitors in blood on PCR detection, researchers have developed various laboratory procedures for sample pretreatment. DNA purification methods suitable for PCR include dialysis, Chelex 100 resin, dilution with cell lysis buffer, centrifugation recovery, washing with NaOH, and addition of bovine serum albumin. However, these pretreatment steps are time-consuming and tedious, requiring different methods for different samples. Pretreatment also results in the loss of valuable nucleic acid and cannot completely remove inhibitors. Adding additives to the PCR system is one strategy to improve PCR results in complex samples, minimizing the impact of PCR inhibitors. For example, the addition of bovine serum albumin or single-stranded DNA binding proteins can enhance the amplification ability of certain polymerases.

[0040] Modifying the DNA polymerase itself is also a viable strategy for improving its ability to perform PCR in complex samples. For some complex samples, using a polymerase with good inhibition tolerance can avoid sample pretreatment steps, saving labor, avoiding cross-contamination, and enabling direct amplification of complex samples. These DNA polymerase mutants are tolerant to common inhibitory substances such as heme, humic acid, or tannic acid. Using these DNA polymerases, direct PCR can be performed on DNA from samples containing these inhibitory substances, reducing the time and cost required for pretreatment and the resulting loss and damage to DNA.

[0041] Those skilled in the art can flexibly add commonly used elements from the prior art to the aforementioned DNA polymerase mutants. Such elements include, but are not limited to, promoters for regulating transcription and translation, molecular tags for protein purification, signal peptides for protein localization, and other known protein sequences from the prior art. Such elements do not affect the activity of the DNA polymerase mutants, including, but not limited to, one or more of polymerization ability, inhibition resistance, or multiplex PCR capability.

[0042] The aforementioned amino acid mutations were experimentally investigated in the examples of this application and all exhibited DNA polymerase activity compared to the parent amino acid sequence of SEQ ID NO: 1. Proteins having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or greater homology to the aforementioned amino acid sequence and having the same ability to enhance enzyme activity are also provided.

[0043] In this specification, homology refers to the "identity" between amino acid sequences, that is, the total ratio of identical amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0044] A protein having 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology with the above-mentioned protein and having the same function, wherein the active site, active pocket, active mechanism, protein structure, etc. are most likely the same as those of the above-mentioned protein, and the protein is a homologous protein obtained by amino acid mutation.

[0045] Sequences with the aforementioned homology can be obtained by amino acid substitution or replacement. Substitution or replacement rules generally apply, and amino acids with similar properties generally have similar effects when substituted with each other. For ease of description, the amino acid residue abbreviations are listed below: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0046] Amino acid substitutions or replacements, for example, in the above homologous proteins, conservative amino acid substitutions may occur. "Conservative amino acid substitutions" include but are not limited to:

[0047] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0048] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0049] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0050] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0051] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0052] In a second typical embodiment of the present application, a DNA molecule is provided, which comprises a polynucleotide encoding the above-mentioned DNA polymerase mutant.

[0053] Preferably, the DNA molecule comprises the nucleotide sequence shown in SEQ ID NO:2.

[0054] SEQ ID NO: 2:

[0055] Furthermore, those skilled in the art can flexibly add nucleic acid sequences for expressing commonly used elements in the prior art near the above-mentioned DNA molecules, including but not limited to promoters for regulating transcription and translation levels, molecular tags for purifying proteins, signal peptides for localizing proteins, and other known sequences in the prior art.

[0056] In a third typical embodiment of the present application, a recombinant vector is provided, which contains the above-mentioned DNA molecule.

[0057] The DNA can encode the DNA polymerase mutant and can be linked to a recombinant vector to form a circular DNA. Both the DNA and the recombinant vector can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the DNA polymerase mutant. For different host species of the DNA molecule or recombinant vector, the nucleotide sequence can be flexibly codon-optimized using existing technologies to obtain a nucleotide sequence with higher transcription and translation efficiency.

[0058] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned DNA molecule or recombinant vector.

[0059] In a preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0060] The above-mentioned host cells can be used to replicate recombinant vectors and transcribe and translate DNA molecules carried by the recombinant vectors, thereby obtaining a large number of DNA polymerase mutants. DNA polymerase mutants can be obtained by disrupting the host cells, purifying proteins after disruption, or other methods using existing technologies. The host cells are not of plant or animal origin and are not developmentally totipotent.

[0061] In a fifth typical embodiment of the present application, a PCR kit is provided, which includes the above-mentioned DNA polymerase mutant.

[0062] In a preferred embodiment, the PCR kit further includes any one or more of the following components: 1) a buffer for providing a PCR amplification environment; 2) PCR primers; 3) a reagent for extracting target DNA.

[0063] The above-mentioned PCR primers include but are not limited to universal primers for amplifying specific target DNA, including but not limited to 16S universal amplification primers for prokaryotic bacteria, 18S universal amplification primers for eukaryotic bacteria, ITS universal amplification primers for fungi, or other PCR primers designed for specific organisms or specific target fragments.

[0064] The aforementioned buffer is a common PCR buffer in the prior art and is used to provide reaction conditions suitable for the aforementioned DNA polymerase mutant. The DNA polymerase mutant of the present application is similar to wild-type Taq DNA polymerase and can perform PCR reactions in the PCR buffer of the prior art. Those skilled in the art can also flexibly optimize the components of the aforementioned PCR buffer to obtain a buffer more suitable for such a DNA polymerase mutant.

[0065] The above reagents for extracting target DNA can extract DNA from the target sample and provide an amplification template for the subsequent PCR reaction.

[0066] In a sixth typical embodiment of the present application, a PCR method is provided, which comprises: performing PCR amplification on the target DNA using the above-mentioned DNA polymerase mutant or the above-mentioned kit.

[0067] In a preferred embodiment, PCR amplification includes multiplex PCR amplification; preferably, the PCR system contains a PCR inhibitor; more preferably, the PCR inhibitor includes but is not limited to one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions and iron ions.

[0068] Utilizing any of the aforementioned DNA polymerase mutants, the aforementioned kits, and the aforementioned PCR methods, PCR reactions can be performed on reaction systems containing multiple inhibitors.

[0069] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0070] Plasmids encoding different DNA polymerase mutant sequences were constructed by site-directed mutagenesis PCR, as detailed in Example 1. These plasmids encoding different DNA polymerase mutant sequences were then transferred into E. coli for culture and induced expression. Finally, the DNA polymerases were purified by heat treatment and affinity chromatography to obtain Taq DNA polymerases containing different mutations, as detailed in Example 2. The resulting mutants were tested for polymerase activity, inhibition resistance, and multiplex PCR, yielding superior mutants with improved performance. Furthermore, the superior mutation sites were combined and superimposed to obtain combined mutants with improved performance.

[0071] By modifying the wild-type Taq DNA polymerase, a superior mutant was obtained, whose enzyme activity, inhibition resistance, and multiplex PCR capabilities were significantly improved compared to the wild-type. The increased polymerase activity can increase the polymerization rate, rapidly obtaining large quantities of product; the improved inhibition resistance allows for direct amplification of complex templates without the need for template pretreatment, saving steps and simplifying operation; and the improved multiplex amplification capability allows for the simultaneous amplification of multiple targets in a single round of PCR, improving efficiency.

[0072] Example 1 Construction of Taq DNA polymerase and its mutant expression plasmids

[0073] The specific implementation steps are as follows:

[0074] (1) Construction of wild-type Taq DNA polymerase expression plasmid

[0075] The gene sequence of wild-type Taq DNA polymerase is shown in SEQ ID NO: 2, and the amino acid sequence encoded by it is shown in SEQ ID NO: 1.

[0076] SEQ ID NO: 2:

[0077] SEQ ID NO: 1:

[0078] The expression plasmid pET-28a(+)-Taq, containing the coding sequence for wild-type Taq DNA polymerase, was synthesized and constructed by Beijing Liuhe BGI Genomics Co., Ltd. Six histidine residues (6×His) were fused to the N-terminus of the amino acid sequence to facilitate protein purification.

[0079] (2) Construction of mutant Taq DNA polymerase expression plasmid

[0080] Forward and reverse mutation primer pairs were designed based on the mutation sites screened by rational design. The primer sequences are shown in Table 1 below.

[0081] Table 1

[0082] The constructed single point or combination mutants are shown in Table 2 below.

[0083] Table 2

[0084] Single-site mutants were introduced into site-directed mutations by PCR amplification using Q5 High-Fidelity DNA Polymerase (purchased from New England Biolabs, catalog number M0491V) on the basis of wild-type Taq DNA polymerase expression plasmid using the above primer pairs. The specific reaction system is shown in Table 3 below.

[0085] Table 3

[0086] The PCR reaction conditions are shown in Table 4 below.

[0087] Table 4

[0088] After the reaction, 1 μL of DpnI (NEB, Catalog No. R0176V) was added and digested at 37°C for 2 h. 5 μL of the digested product was then transformed into E. coli competent DH5α cells (purchased from Tiangen Biotechnology Co., Ltd., Catalog No. CB101). A single colony was picked from the plate and cultured, and the plasmid was extracted. The plasmid was then sent to Beijing Liuhe BGI Genomics Co., Ltd. for Sanger sequencing, and the correct mutant was analyzed by sequencing comparison.

[0089] Combination mutations refer to mutants with two or more mutations relative to wild-type Taq DNA polymerase. Combination mutations are constructed by first performing site-directed mutagenesis at one mutation site using corresponding primers. Once the mutant product is obtained, a second site-directed mutagenesis is performed on the mutant product, and so on until multiple mutations are obtained relative to wild-type Taq DNA polymerase. The PCR reaction system and reaction conditions are as described above. For some primers, the annealing temperature can be adjusted based on the primer Tm value.

[0090] Example 2 Inducible expression and purification of Taq DNA polymerase and its mutants

[0091] The wild-type and mutant plasmids were transformed into BL21 (DE3) competent cells (purchased from Tiangen Biotechnology Co., Ltd., product number CB105), and then single colonies were picked and inoculated into 2 mL of LB medium containing kanamycin resistance (50 μg / mL) and cultured overnight at 37°C at 200 rpm / min. The next day, 200 μL of the bacterial solution was transferred to 10 mL of LB medium containing kanamycin resistance (50 μg / mL) and cultured at 37°C at 200 rpm / min until the OD 600 At 0.6-0.8, the inducer IPTG was added to a final concentration of 0.5 mM and cultured at 37°C and 200 rpm / min for 4 hours to induce expression. The culture was finally centrifuged at 8000 rpm / min for 5 minutes to collect the induced bacteria.

[0092] Resuspend the collected cells in cell resuspension buffer (50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, pH 7.8). Add 100 μL of 10 mg / mL lysozyme to the resuspended cells and incubate at 37°C for 30 minutes. Heat the mixture at 75°C for 40 minutes. Centrifuge at 14,000 rpm for 10 minutes, and collect the supernatant.

[0093] Purification was performed using a His SpinTrap column (Cytiva, Catalog No. 28401353). 500 μL of the supernatant was added to the His SpinTrap column, centrifuged at 100 g for 30 seconds, and the filtrate was discarded. An equal volume of Wash Buffer 1 (50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, 30 mM Imidazole, pH 7.8) was added, and the column was centrifuged at 100 g for 30 seconds. The filtrate was discarded. An equal volume of Wash Buffer 2 (50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, 50 mM Imidazole, pH 7.8) was added, and the column was centrifuged at 100 g for 30 seconds. The filtrate was discarded. Elution was performed twice with 200 μL of Elution Buffer (50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, 500 mM Imidazole, pH 7.8). The eluates were then concentrated using a 0.5 mL 50 kDa ultrafiltration tube (Millipore, Cat. No. UFC505096). Centrifuge at 7000 rpm for 5 min and discard the filtrate. Add 500 μL of Dialysis Buffer (40 mM Tris-HCl, 200 mM KCl, 2 mM DTT, 0.2 mM EDTA-Na2, 5% Glycerol, pH 7.8) and centrifuge at 7000 rpm for 3-5 min. Discard the filtrate. Repeat this process 5-6 times and collect the enzyme solution in the tube. After protein quantification, prepare an enzyme stock (containing 50% Glycerol) at a stock concentration of 0.1 mg / mL. The hot start antibody was then prepared into a concentration of 0.05 mg / mL for subsequent determination and analysis. For details on the hot start antibody and its use, refer to patent application CN114685671A.

[0094] Example 3 Determination and analysis of polymerization activity of Taq DNA polymerase and its mutants

[0095] The polymerase activity of the purified wild-type Taq DNA polymerase and its mutants was determined. The activity was determined using primer-bound M13 ssDNA (NEB, Catalog No. N4040S) as a template-primer complex. Under the action of the DNA polymerase, the DNA chain was extended to produce an extended double-stranded DNA product. The activity of the DNA polymerase was calculated by detecting the amount of double-stranded DNA generated. The sequence of the bound primer was: 5'-AGCGAACCTCCCGACTTGCGGGAGG-3' (SEQ ID NO: 65). The formula of the 10× PCR buffer used in the present invention was: 100mM Tris-HCl, 500mM KCl, 15mM MgCl2, 25% Glycerol, and 0.5mg / mL BSA (bovine serum albumin).

[0096] The reaction system for Taq DNA polymerase activity detection is shown in Table 5 below.

[0097] Table 5

[0098] The prepared reaction system was placed in a PCR instrument and reacted at 72°C for 5 min. After that, 0.5 μL of 0.5 M EDTA was added to terminate the reaction. The amount of double-stranded DNA was then detected using the Qubit dsDNA HS Assay Kit (purchased from Thermo Fisher Scientific, catalog number Q32854). The polymerization activity of the mutant relative to the wild type was calculated (the polymerization activity of the wild type was set as 1) as shown in Table 6.

[0099] Table 6

[0100] Activity test results showed that the relative enzyme activity value greater than 1 indicated that the polymerization activity of the mutant Taq DNA polymerase was improved compared with the wild-type Taq DNA polymerase. Mutants Mut1 (G59W), Mut4 (D488K), Mut5 (G59W + D488K), Mut6 (S543T), Mut8 (E388K), Mut9 (E400R), Mut10 (E397Q), Mut12 (L549F), Mut17 (T664F), Mut19 (G59W + D488K + L549F), Mut21 (G59W + S577T), Mut22 (G59W + E388K), Mut3 The polymerization activities of ut23 (G59W+E400R), Mut27 (E400R+D488K), Mut30 (D488K+L549F), Mut38 (E388K+E397Q), Mut42 (E388K+T664F), Mut43 (S543T+T664F), Mut45 (E400R+T664F), Mut58 (L678I), and Mut63 (E388K+L549F) were improved to varying degrees compared with the wild type.

[0101] Example 4 Multiplex PCR testing and analysis of Taq DNA polymerase and its mutants

[0102] In order to obtain DNA polymerase mutants that can be used for multiplex PCR amplification, the purified wild-type Taq DNA polymerase and its mutants were subjected to eight-fold human housekeeping gene amplification tests.

[0103] The template for 8-plex amplification was human genomic DNA (human gDNA), which was extracted from Expi293F using a DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd., catalog number DP304-02). TM The fragments were extracted from cells (purchased from Thermo Fisher Scientific, catalog number A14527). The amplification primers and target fragment sizes are shown in Table 7 below.

[0104] Table 7

[0105] The PCR reaction system for 8-plex amplification of human housekeeping genes is shown in Table 8 below.

[0106] Table 8

[0107] The PCR conditions for 8-plex amplification of human housekeeping genes are shown in Table 9 below.

[0108] Table 9

[0109] After the 8-plex amplification of the human housekeeping gene is completed, 5 μL of 6x DNA Loading Buffer (60mM Tris-HCl, 60mM EDTA, 60% Glycerol, 0.15% (m / V) OrangeG) is added and mixed thoroughly. Then, electrophoresis analysis is performed on a 2% agarose gel to select mutants that can amplify all bands, or have more bands than the wild type, or have increased yield, or improved uniformity. Some bands in the figure have tailing due to the presence of certain non-specific amplification phenomena, which has nothing to do with the multiplex amplification ability of the DNA polymerase and can be ignored. Specific results are shown in Figures 1, 2, and 3. In the figures, WT represents the band corresponding to the wild-type DNA polymerase (SEQ ID NO: 1), and M is the marker band.

[0110] The results in Figures 1, 2, and 3 show brighter or more amplified bands in agarose gel electrophoresis, indicating that the mutant DNA polymerases have enhanced multiplex amplification capabilities compared to the wild-type DNA polymerase. Mut5 (G59W+D488K), Mut6 (S543T), Mut9 (E400R), Mut11 (T544Y), Mut12 (L549F), Mut19 (G59W+D488K+L549F), Mut20 (G59W+S543T), Mut21 (G59W+S577T), and Mut23 (G59W+E400R) significantly improved amplification performance when used in multiplex PCR.

[0111] Example 5 Inhibition resistance test of Taq DNA polymerase and its mutants

[0112] To identify DNA polymerase mutants with improved inhibition resistance, purified wild-type Taq DNA polymerase and its mutants were tested in PCR in an inhibitor-containing system. The PCR system contained inhibitors (12 μM heme, 1.6 μg / mL humic acid, or 1.2 μg / mL tannic acid). The specific components of the PCR system are described in Table 10 below.

[0113] Table 10

[0114] The PCR reaction conditions are shown in Table 11 below.

[0115] Table 11

[0116] After amplification, add 5 μL of 6x DNA Loading Buffer, mix thoroughly, and analyze by electrophoresis on a 2% agarose gel to identify mutants that amplify the target band. Detailed results are shown in Figure 4. Two sets of experiments were performed on the WT: one without inhibitor and one with inhibitor, as with the other mutants. The presence of tailing in some bands in the figure is due to nonspecific amplification and is not related to the inhibition tolerance of the DNA polymerase and is not a concern.

[0117] The results in Figure 4 show that the appearance of amplified bands in the agarose gel electrophoresis diagram indicates that the mutant Taq DNA polymerase has enhanced inhibition resistance compared to the wild-type Taq DNA polymerase. Mutants Mut1 (G59W), Mut5 (G59W+D488K), Mut6 (S543T), Mut7 (S577T), Mut9 (E400R), Mut12 (L549F), Mut17 (T664F), Mut18 (G59W+D488K+S543T), Mut19 (G59W+D488K+L549F), Mut21 (G59W+S577T), Mut22 (G59W+E388K), Mut43 (S543T+T664F), Mut58 (L678I), and Mut63 (E388K+L549F) have increased tolerance to at least one inhibitor. Among them, Mut5 (G59W+D488K), Mut19 (G59W+D488K+L549F), Mut21 (G59W+S577T), and Mut22 (G59W+E388K) all showed improved tolerance to the three inhibitors.

[0118] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0119] The Taq DNA polymerase mutant disclosed in this application has enhanced polymerization activity and inhibitor tolerance, enabling PCR reactions with templates containing inhibitory substances, eliminating the need for template extraction, purification, and other pre-PCR treatments. These DNA polymerase mutants are also capable of performing multiplex PCR. Using these DNA polymerase mutants, more products can be obtained in the same amount of time, or PCR reactions with templates containing inhibitory substances can be performed without the need for template extraction, purification, and other pre-PCR treatments, or multiplex PCR can be performed. Compared to the wild type, these mutants exhibit significantly improved DNA polymerization activity, inhibitor tolerance, and multiplex amplification.

[0120] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A DNA polymerase mutant, characterized in that, The DNA polymerase mutant has DNA polymerase activity and includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology with the amino acid sequence shown in SEQ ID NO:

1.

2. The DNA polymerase mutant according to claim 1, wherein The DNA polymerase mutant has an amino acid substitution at at least one of the following sites relative to the amino acid sequence shown in SEQ ID NO: 1: D488, S577, S543, E388, E400, E397, T544, L549, V586, L670, L678, T664 and G59.

3. The DNA polymerase mutant according to claim 2, wherein the amino acid substitution at the D488 site includes D488H, D488R or D488K; the amino acid substitution at the S543 site includes S543T; the amino acid substitution at the S577 site includes S577T; the amino acid substitution at the E388 site includes E388K; the amino acid substitution at the E400 site includes E400R; the amino acid substitution at the E397 site includes E397Q; the amino acid substitution at the T544 site includes T544Y, T544D, T544K, T544S, T544W or T544F; the amino acid substitution at the L549 site includes L549F, L549I, L549D or L549K; the amino acid substitution at the V586 site includes V586F; the amino acid substitution at the L670 site includes L670F; the amino acid substitution at the L678 site includes L678Y, L678W, L678I, L678F, L678K or L678D; the amino acid substitution at the T664 site includes T664F; the amino acid substitution at the G59 site includes G59W.

4. The DNA polymerase mutant according to claim 3, wherein The mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: D488H, D488R, D488K, S577T, G59W, E388K, E397Q, E400R, S543T, T544Y, T544D, T544K, T544S, T544W, T544F, L549F, L549I, L549D, L549K, V586F, T664F, L670F, L678Y, L678K, L678D, L678I, L678F, G59W+E388K, G59W+E400R, G59W+D488K, G59W+S543T, G59W+S577T, E388K+E397Q, E388K+E400R, E388K+D488K, E388K+S543T, E388K+T544Y, E388K+L549F, E388K+S577T, E388K+L670F, E388K+T664F, E388K+L678Y, E397Q+D488K, E397Q + T664F, E400R + D488K, E400R + T664F, D488K + S543T, D488K + T544Y, D488K + L549F, D488K + S577T, D488K + V586F, D488K + T664F, D488K + L670F, D488K + L678Y, S543T + T664F, S577T + T664F, V586F + T664F, T664F + L670F, T664F + L678W, T664F + L678Y, G59W + D488K + S543T or G59W + D488K + L549F; Preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W, D488K, G59W + D488K, S543T, E388K, E400R, E397Q, L549F, T664F, G59W + D488K + L549F, G59W + S577T, G59W + E388K, G59W + E400R, E400R + D488K, D488K + L549F, E388K + E397Q, E388K + T664F, S543T + T664F, E400R + T664F, L678I or E388K + L549F, and the DNA polymerase activity of the DNA polymerase mutant is superior to the DNA polymerase activity of the amino acid sequence shown in SEQ ID NO: 1; Preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W + D488K, S543T, E400R, T544Y, L549F, G59W + D488K + L549F, G59W + S543T, G59W + S577T or G59W + E400R, and the multiplex amplification ability of the DNA polymerase mutant is superior to the multiplex amplification ability of the amino acid sequence shown in SEQ ID NO: 1; Preferably, the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W, G59W + D488K, S543T, S577T, E400R, L549F, T664F, G59W + D488K + S543T, G59W + D488K + L549F, G59W + S577T, G59W + E388K, S543T + T664F, L678I, E388K + L549F, G59W + D488K, G59W + D488K + L549F, G59W + S577T or G59W + E388K, and the PCR inhibitor tolerance ability of the DNA polymerase mutant is superior to the PCR inhibitor tolerance ability of the amino acid sequence shown in SEQ ID NO:

1.

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

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

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

8. The host cell according to claim 7, wherein, The host cell includes a prokaryotic cell or a eukaryotic cell; Preferably, the prokaryotic cell includes Escherichia coli.

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

10. The PCR kit according to claim 9, characterized in that, The PCR kit further includes any one or more of the following components: 1) A buffer for providing a PCR amplification environment; 2) PCR primers; 3) A reagent for extracting the target DNA.

11. A PCR method, characterized in that, The PCR method includes: performing PCR amplification on the target DNA by using the DNA polymerase mutant according to any one of claims 1 to 4 or the PCR kit according to claim 9 or 10.

12. The PCR method according to claim 11, wherein The PCR includes multiplex PCR amplification; Preferably, the PCR system contains a PCR inhibitor; More preferably, the PCR inhibitor includes one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions, and iron ions.

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