A DNA polymerase mutant and application thereof

Abstract

The application discloses a Taq-DNA polymerase mutant without 5' to 3' exonuclease activity and application thereof.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically to a mutant of Taq DNA polymerase and its applications. Background Technology

[0002] Polymerase chain reaction (PCR) is a widely used in vitro DNA amplification technique. Since its introduction in 1985, it has gradually expanded into various fields, playing a vital role not only in laboratory molecular biology but also in clinical disease diagnosis and forensic identification. For example, PCR can be used to detect gene mutations and microbial or viral infectious agents, as well as antibiotic resistance genes and biohazards. One of the earliest diagnostic applications of PCR was for prenatal diagnosis of sickle cell anemia, where PCR detection of sickle cell mutations was faster and more sensitive than previous methods. Subsequently, more PCR-based diagnostic methods were developed, including: diagnosing viral (such as HIV) infection by detecting low-copy-number viral targets; diagnosing tuberculosis by detecting Mycobacterium tuberculosis; and diagnosing Helicobacter pylori infection by detecting different isolates of Helicobacter pylori from the gastrointestinal tract. In 1992, Higuchi et al. developed real-time PCR, an enhanced PCR method that uses fluorescent dyes (such as SYBR Green I) or fluorescence resonance energy transfer (FRET) probes to detect the amount of product formed during the reaction in real time. Compared with traditional culture methods, PCR-based detection methods have advantages such as high speed, high specificity, and high sensitivity.

[0003] PCR reactions primarily rely on DNA polymerases. Initially, the enzymes used to amplify DNA in PCR technology were derived from the Klenow fragment of E. coli polymerase, which could produce billions of copies of molecules in just a few hours. Subsequently, several highly thermostable DNA polymerases were isolated from thermophilic bacteria, including *Thermus aquaticus* (Taq), *Thermus thermophilus* (Tth), and *Pyrococcus furiosus* (Pfu), which retain their activity even at 95°C. Taq DNA polymerase was the first enzyme used in PCR, exhibiting both polymerase and exonuclease activity. Due to its high stability, high efficiency, and simple and economical production process, Taq DNA polymerase is the most popular and widely used enzyme in most PCR applications.

[0004] Taq DNA polymerase possesses both 5' to 3' polymerase activity and 5' to 3' exonuclease activity (also referred to as "exonuclease activity" in this paper). Furthermore, it has been found to possess pyrophosphatase activity, catalyzing the reaction of ddNMP and PPi to generate ddNTPs. Taq DNA polymerase also exhibits template-independent activity, allowing the introduction of a non-template complementary nucleotide A at the 3' end of the DNA molecule, resulting in a PCR product with a single nucleotide A protruding at the 3' end. Taq DNA polymerase has been shown to possess reverse transcription activity and can be combined with Pfu DNA polymerase to obtain cDNA from eukaryotic genes. Moreover, compared to commonly used avian AMV reverse transcriptase and murine Mulv reverse transcriptase, reverse transcription using Taq DNA polymerase at high temperatures avoids the adverse effects of mRNA secondary structure.

[0005] Taq DNA polymerase possesses 5' to 3' exonuclease activity, enabling it to hydrolyze the DNA strand leading up to the DNA growth chain from the 5' to 3' direction, primarily producing 5'-deoxynucleotides. This enzymatic activity is only effective at cleaving phosphodiester bonds in paired DNA. This can lead to fragmentation of PCR products, affecting PCR amplification. In genetic engineering, DNA sequencing is frequently required. Compared to conventional sequencing enzymes, Taq DNA polymerase offers advantages in DNA sequencing, including excellent strand extension performance and the ability to operate at high temperatures. This allows it to overcome the impact of GC-rich templates forming their own secondary structures on sequencing. When PCR is combined with end-terminated sequencing, only a small amount of template is needed, and sequencing of double-stranded templates does not require alkaline denaturation. The thermostability of Taq DNA polymerase allows for annealing at high temperatures during PCR, enhancing the specificity of primer-template binding.

[0006] Fluorescently labeled nucleotides have greatly simplified and improved the efficiency of many procedures in molecular biology. In synthesis, the use of fluorescently labeled nucleotides to label polynucleotides has largely replaced the use of radioactive labeling. However, a major problem with the use of fluorescently labeled nucleotides is the ability of DNA polymerases to distinguish the incorporation of fluorescently labeled nucleotides. It has been found that in competitive analysis between TET (6-carboxy-47.2,7'-TET rachlorofluresein)-labeled 2',3'-dideoxynucleotides and their corresponding unlabeled dideoxynucleotides, Taq DNA polymerase incorporates unlabeled dideoxynucleotides into DNA at least 85 times more frequently than their corresponding labeled nucleotides. This distinction between labeled and unlabeled nucleotides has profound implications for procedures using DNA polymerase to label DNA. For example, large quantities of fluorescently labeled nucleotides must be used in sequencing reactions. These large quantities of fluorescently labeled nucleotides are expensive and generate excessive background fluorescence, thus reducing the yield of sequence information. This ability of DNA polymerases to distinguish fluorescently labeled nucleotides adversely affects many molecular biology procedures that require the enzymatic addition of fluorescently labeled nucleotides, such as dideoxy-labeled terminator sequencing. In addition, Taq DNA polymerase has different preferences for various ddNTPs when used for sequencing, which makes the incorporation rate of the four ddNTPs significantly different. Among them, the incorporation rate of ddGTP is 10 times faster than that of ddATP, ddCTP and ddTTP. This results in an imbalance in the intensity and peak height of the four bands during sequencing, which is not conducive to the interpretation of results and affects the accuracy of sequencing results.

[0007] In addition, with the continuous advancement of research, many methods for detecting rare mutations have been developed, such as single-strand conformation polymorphism (SSCP), heteroduplex technology (HA), denaturing high-performance liquid chromatography (DHPLC), denaturing gradient gel electrophoresis (DGGE), chemical mismatch lysis (CMC), mass spectrometry, DNA microarray technology, and pyrophosphate-activated polymerase reaction (PAP). Pyrophosphate-activated polymerase reaction is currently one of the most effective techniques for detecting rare mutations. DNA polymerases possess not only polymerization activity but also pyrophosphate activity. Polymerase activity enables the dNTP→dNIP+PPi reaction in PCR, while pyrophosphate activity enables the dNMP+PPi→dNTP reaction. A primer with ddNMP-blocked ends is designed; only when the 3' end of the primer is completely paired with the template DNA sequence can the pyrophosphate reaction (dNMP+PPi→ddNTP) occur, causing the ddNMP at the end to detach, thus releasing the block and allowing the primer to extend. If the primers do not match the template, the pyrophosphatase reaction cannot occur, and the primers cannot extend because their 3' ends are blocked. This allows for the differentiation of single nucleotides. However, Taq DNA polymerase has different preferences for various ddNTPs, which affects the accuracy of PAP reaction results.

[0008] In sequencing and PAP (Programmable Angioplasty), a series of problems arise due to the inconsistent ability of DNA polymerases to distinguish between ddNTPs and fluorescently labeled nucleotides. This inconsistency is mainly caused by the excessively high 5' to 3' exonuclease activity of Taq DNA polymerase. Therefore, it is desirable to develop novel Taq DNA polymerases with no 5' to 3' exonuclease activity but excellent polymerase activity for application in various scenarios, such as PAP and sequencing technologies. Summary of the Invention

[0009] Through in-depth research, the inventors of this application have developed a mutant of Taq DNA polymerase that not only possesses excellent polymerase activity (i.e., the ability to obtain large amounts of DNA product in a short time) but also exhibits significantly reduced 5' to 3' exonuclease activity. This mutant is particularly advantageous for clinical diagnostics and reagent kit development, and is especially advantageous for sequencing and PAP reactions.

[0010] Therefore, in one aspect, this application provides a mutant Taq DNA polymerase or a variant thereof, wherein the mutant Taq DNA polymerase has the following mutations compared to the wild-type Taq DNA polymerase:

[0011] (1) The amino acid residue at position 345 of SEQ ID NO:1 is replaced by a valine residue;

[0012] (2) The amino acid residue at position 520 of SEQ ID NO:1 is replaced by a glycine residue; and

[0013] (3) The amino acid residue at position 578 of SEQ ID NO:1 is replaced by an asparagine residue;

[0014] The variant, compared to the mutated Taq DNA polymerase, has at least 90%, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or, has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conserved substitutions), additions, or deletions; and,

[0015] The amino acid residues at positions 345, 520, and 578 of SEQ ID NO:1 in the variant are identical to those in the mutated Taq DNA polymerase; and

[0016] The variant retains the function of the mutated Taq DNA polymerase (e.g., having higher DNA polymerase activity than wild-type Taq DNA polymerase and lower 5' to 3' exonuclease activity than wild-type Taq DNA polymerase).

[0017] The mutant Taq DNA polymerase or its variants of this application possess excellent DNA polymerase activity. For example, the polymerase activity of the mutant Taq DNA polymerase or its variants of this application is approximately at least 2, 3, 4, 5, or 6 times that of the wild-type Taq DNA polymerase. Furthermore, the mutant Taq DNA polymerase or its variants of this application also exhibit significantly reduced 5' to 3' exonuclease activity. For example, the exonuclease activity of the mutant Taq DNA polymerase or its variants of this application does not exceed approximately 10%, 5%, or 1% of that of the wild-type Taq DNA polymerase. In some preferred embodiments, the mutant Taq DNA polymerase or its variants of this application substantially lack 5' to 3' exonuclease activity.

[0018] In some preferred embodiments, the wild-type Taq DNA polymerase has an amino acid sequence as shown in SEQ ID NO:1.

[0019] In some preferred embodiments, the mutated Taq DNA polymerase has an amino acid sequence as shown in SEQ ID NO:2.

[0020] In another aspect, this application provides an isolated nucleic acid comprising a nucleotide sequence encoding a mutated Taq DNA polymerase or a variant thereof as described above. In some preferred embodiments, the isolated nucleic acid of this application has a nucleotide sequence as shown in SEQ ID NO:3.

[0021] In another aspect, this application provides a vector containing the isolated nucleic acid. Vectors that can be used to insert a target polynucleotide are well known in the art, including but not limited to cloning vectors and expression vectors. In one embodiment, the vector is, for example, a plasmid, a granule, a bacteriophage, etc.

[0022] In another aspect, this application also relates to host cells comprising the isolated nucleic acids or vectors described above. Such host cells include, but are not limited to, prokaryotic cells such as *E. coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell of this invention can also be a cell line, such as 293T cells. In some preferred embodiments, the host cell is *E. coli*.

[0023] In another aspect, this application also relates to compositions comprising the aforementioned mutated Taq DNA polymerase or variants thereof, or the aforementioned isolated nucleic acids, vectors, or host cells. In some preferred embodiments, the compositions comprise the Taq DNA polymerase or variants thereof of the present invention.

[0024] In another aspect, this application relates to a method for preparing a mutant Taq DNA polymerase or a variant thereof as described above, comprising expressing the mutant Taq DNA polymerase or a variant thereof in a host cell and then recovering the mutant Taq DNA polymerase or a variant thereof from a culture of the host cell.

[0025] In some preferred embodiments, the host cell is Escherichia coli.

[0026] In some preferred embodiments, the method includes the steps of: expressing the mutated Taq DNA polymerase or a variant thereof in *E. coli*, and then purifying the mutated Taq DNA polymerase or a variant thereof from the lysate supernatant of the *E. coli*. In some preferred embodiments, the mutated Taq DNA polymerase or a variant thereof is recovered from the lysate supernatant of the *E. coli* by chromatography (e.g., cation exchange chromatography, hydroxyapatite chromatography, and / or hydrophobic interaction chromatography).

[0027] In another aspect, this application also relates to the use of the mutated Taq DNA polymerase or variants thereof for nucleic acid synthesis or amplification (e.g., PCR).

[0028] In another aspect, this application also relates to the use of the mutated Taq DNA polymerase or a variant thereof for a pyrophosphate-activated polymerase reaction (PAP). In some preferred embodiments, the pyrophosphate-activated polymerase reaction (PAP) is used to detect rare mutations.

[0029] In another aspect, this application also relates to the use of the mutated Taq DNA polymerase or a variant thereof for analyzing or determining the nucleotide sequence of a nucleic acid (e.g., DNA) molecule. In a preferred embodiment, the nucleotide sequence analysis or determination process includes the following steps: incubating a primer molecule capable of hybridizing with the nucleic acid molecule with the nucleic acid molecule and the mutated Taq DNA polymerase or a variant thereof; and determining at least a portion of the nucleotide sequence of the nucleic acid molecule.

[0030] In another aspect, this application also relates to a kit containing the mutated Taq DNA polymerase or a variant thereof. The kit of this application can be used for various purposes, such as nucleic acid synthesis or amplification (e.g., PCR), PAP, or sequencing reactions. In some preferred embodiments, the kit further includes reagents selected from: reagents for performing PCR (e.g., buffers, dNTPs, primers); reagents for performing PAP (e.g., buffers, dNTPs, primers, pyrophosphate, or analogs thereof); reagents for performing sequencing reactions (e.g., buffers, dNTPs, primers, synthesis terminators); or any combination thereof.

[0031] Explanation and interpretation of relevant terms in this application

[0032] In this application, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0033] According to the present invention, the term "variant" refers to a protein whose amino acid sequence, compared to the amino acid sequence of the mutant Taq DNA polymerase of the present invention (such as the protein shown in SEQ ID NO: 2), has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conserved substitutions), additions, or deletions, or has at least 90%, 95%, 96%, 97%, 98%, or 99% identity, and retains the function of the mutant Taq DNA polymerase. The mutant Taq DNA polymerase of this application has higher DNA polymerase activity than the wild-type Taq DNA polymerase. For example, the polymerase activity of the mutant Taq DNA polymerase of this application is approximately at least 2, at least 3, at least 4, at least 5, or 6 times that of the wild-type Taq DNA polymerase. Furthermore, the mutant Taq DNA polymerase of this application also has lower 5' to 3' exonuclease activity than the wild-type Taq DNA polymerase. For example, the exonuclease activity of the mutant Taq DNA polymerase of this application does not exceed approximately 10%, 5%, or 1% of that of the wild-type Taq DNA polymerase. In some preferred embodiments, the mutant Taq DNA polymerase of this application substantially lacks 5' to 3' exonuclease activity.

[0034] The term "identity" is a measure of the similarity between nucleotide or amino acid sequences. Sequences are typically arranged to achieve the maximum possible match. "Identity" itself has a well-known meaning in the art and can be calculated using publicly available algorithms (such as BLAST).

[0035] According to the present invention, the term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be achieved using, for example, methods readily available through computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0036] As used herein, the expression "the position corresponding to the Xth bit of SEQ ID NO:1" refers to the position in the target sequence that is equivalent to the Xth bit of SEQ ID NO:1 when the target sequence is compared with SEQ ID NO:1 to produce maximum identity.

[0037] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the essential properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, conservative substitution generally refers to replacing a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0038] This article defines “significantly reduced 5' to 3' exonuclease activity” as: (1) having about or less than 10%, or preferably about or less than 5% or 1%, of the 5' to 3' exonuclease activity of the corresponding unmutated wild-type enzyme (e.g., wild-type Taq DNA polymerase).

[0039] According to the present invention, the term "vector" refers to a nucleic acid delivery vehicle in which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, etc.

[0040] According to the present invention, the term "lysis supernatant" refers to a solution produced by the following steps: lysing host cells (e.g., *E. coli*) in a lysis buffer, and then removing insoluble matter from the lysis buffer containing the lysed host cells. Various lysis buffers are well known to those skilled in the art, including but not limited to Tris buffer, phosphate buffer, HEPES buffer, MOPS buffer, etc. Furthermore, host cell lysis can be achieved by various methods well known to those skilled in the art, including but not limited to homogenization, sonication, grinding, high-pressure extrusion, lysozyme treatment, etc. Methods for removing insoluble matter from the lysis buffer are also well known to those skilled in the art, including but not limited to filtration and centrifugation.

[0041] In this article, "expression" refers to the method of producing polypeptides from structural genes. It includes transcribing the gene into messenger RNA (mRNA) and translating this mRNA into a polypeptide.

[0042] In this article, "polymerase reaction activated by pyrophosphate hydrolysis" refers to the process by which polymerase uses its pyrophosphatase activity to pyrophosphate the ddNMPs blocked at the ends of primers to produce ddNTPs, causing the ddNMPs at the ends to detach, the primer block is released, and the primer can be extended, allowing the DNA polymerization reaction to continue.

[0043] Beneficial effects of the invention

[0044] (1) The mutant Taq DNA polymerase of this application (e.g., the Taq 07 mutant) or its variants have excellent polymerase activity, which is at least 2, at least 3, at least 4, at least 5, or 6 times that of wild-type Taq DNA polymerase.

[0045] (2) The mutant Taq DNA polymerase of this application (e.g., the Taq 07 mutant) or its variants have significantly reduced 5' to 3' exonuclease activity (e.g., less than about 10%, 5%, or 1% of the 5' to 3' exonuclease activity of wild-type Taq DNA polymerase). For example, the Taq 07 mutant of this application substantially lacks 5' to 3' exonuclease activity.

[0046] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0047] Figure 1This diagram illustrates the principle of detecting the 5' to 3' exonuclease activity of Taq DNA polymerase using a fluorescent probe. The fluorescent probe used is an oligonucleotide probe, with a fluorescent group (e.g., FAM) at its 5' end and a quencher group (e.g., BHQ) downstream of the 5' end. This probe maintains a stable hairpin structure at 55°C. At the start of the reaction, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. As the reaction proceeds, Taq DNA polymerase moves along the 3' end. When it reaches the fluorescent group at the 5' end of the probe, the 5' to 3' exonuclease activity of Taq DNA polymerase cleaves the probe, separating the reporter and quencher groups, allowing the fluorescence monitoring system to receive the fluorescent signal. Therefore, during the reaction, one fluorescent molecule is formed for each DNA strand amplified, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the fluorescent product.

[0048] Figure 2 This diagram illustrates the principle of detecting Taq DNA polymerase activity using Picogreen fluorescent dye. Picogreen is a highly sensitive fluorescent dye that inserts into DNA molecules. At 72°C, the extension of the primers by Taq DNA polymerase is accompanied by the insertion of the fluorescent dye, thus providing real-time reporting of DNA product generation.

[0049] Figure 3 To utilize Figure 1 and Figure 2 The detection system shown plots standard curves of polymerase activity and 5' to 3' exonuclease activity of wild-type Taq DNA polymerase under different concentration conditions. These curves can be used to quantitatively calculate the polymerase activity and 5' to 3' exonuclease activity of other mutants.

[0050] Figure 4 The relative polymerase activities of WT (wild-type), mutant E507K, and Taq07, quantified using wild-type Taq DNA polymerase as a standard. Figure 4 A) and relative 5' to 3' exonuclease activity ( Figure 4 B).

[0051] Sequence information

[0052] Information about the sequence involved in this invention is provided in Table 1 below.

[0053] Table 1

[0054]

[0055]

[0056] Detailed Implementation

[0057] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0058] Unless otherwise specified, the molecular biology experimental methods used in this application are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; enzymes are used according to the conditions recommended by the product manufacturer. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed in this application.

[0059] The wild-type Taq-DNA polymerase used in this embodiment, and the amino acid sequences of the polymerase mutants E507K and Taq07 are shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 2, respectively. The preparation methods of the three polymerases are as follows:

[0060] 1. Pick the bacterial strain containing DNA encoding polymerase and place it in 5 mL of LB medium, incubate overnight at 37°C;

[0061] 2. Transfer the seed culture at a ratio of 1:100 to 500 mL of fresh culture medium and wait for OD... 600 When the concentration reaches between 0.4 and 0.6, add IPTG to a final concentration of 0.2 mM and induce overnight at 37°C;

[0062] 3. Pour the bacterial culture into a 500mL centrifuge bottle, centrifuge at 2500g at 4℃ for 20min, discard the supernatant, and place in a -80℃ refrigerator for 30min;

[0063] 4. Resuspend the bacterial cells in 30 mL of pre-cooled banding buffer (β-ME 1:1000, PMSF 1:100) (if the strain does not contain lysozyme, you can add lysozyme to a final concentration of 0.1 mg / mL).

[0064] 5. Sonicate the bacterial suspension in an ice-water mixture at 6Ω power at 50% for 10-30 minutes (program set to pause for 3 seconds after every 2 seconds of sonication);

[0065] 6. Heat-treat the sonicated suspension in a 75°C water bath for 30 minutes;

[0066] 7. Centrifuge the heated suspension at 12,000 rpm and 4℃ for 20 min to remove cell debris, collect the supernatant S, and take out 100 μL for sample preparation;

[0067] 8. Take 1-2 mL of Ni-NTA Agarose (purchased from QIAGEN, catalog number: 30210), first rinse off the ethanol with 15 mL of double-distilled water, then elute with 5 mL of Elution buffer, 5 mL of Wash buffer, and 15 mL of Banding buffer respectively (each mL of packing can bind approximately 20-30 mg of protein).

[0068] 9. Mix the supernatant from centrifugation in step 7 thoroughly with the beads, incubate at 4°C for at least 1 hour, then allow the liquid to flow out naturally, collect the breakthrough fluid (FT), and take out 100 μL for sample preparation;

[0069] 10. Wash Ni-NTA Agarose with 15 volumes of banding buffer, then allow the liquid to flow out naturally, collect eluent B, and take out 100 μL for sample preparation;

[0070] 11. Wash Ni-NTA Agarose with 1 volume of wash buffer, collect the eluent W1, and take 100 μL for sample preparation; repeat the washing once, collect the eluent W2, and take 100 μL for sample preparation;

[0071] 12. Wash Ni-NTA Agarose with 0.5 volumes of elution buffer, collect eluent E1, and take 100 μL for sample preparation; repeat the elution four times, collect eluent E2, E3, and E4 respectively, and take 100 μL for sample preparation from each.

[0072] 13. The collected S, FT, B, W1, W2, E1, E2, E3, and E4 fractions were stored at 4°C. 100 μL of each fraction was taken out and used for sample preparation. The fraction containing polymerase was determined by SDS-PAGE analysis. The corresponding fractions were then dialyzed overnight to obtain purified polymerase.

[0073] Example 1: Detection of 5' to 3' exonuclease activity and polymerase activity of wild-type Taq DNA polymerase (WT)

[0074] 1. Detection of 5' to 3' exonuclease activity of wild-type Taq DNA polymerase

[0075] Design a single-stranded DNA strand capable of forming a double-ended hairpin structure, which can form a stable hairpin structure during elongation. Design a fluorescent probe within this hairpin structure that can bind to the single-stranded DNA. The 5' nucleotide of this probe carries a FAM fluorescent group, and the downstream nucleotide at the 5' end carries a BHQ quencher group (e.g., ...). Figure 1 As shown, the probe maintains a stable hairpin structure even at 55°C. Before Taq DNA polymerase begins extension, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. As the extension reaction proceeds, Taq DNA polymerase moves along the 3' end. When it reaches the fluorophore at the 5' end of the probe, the 5' to 3' exonuclease activity of Taq DNA polymerase cleaves the probe, separating the reporter and quencher fluorophores, allowing the fluorescence monitoring system to receive the fluorescent signal. Therefore, during the reaction, one fluorescent molecule is formed for each DNA strand amplified, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the fluorescent product. Thus, by detecting the intensity of the fluorescent signal, the level of the 5' to 3' exonuclease activity of the DNA polymerase can be determined. The sequence of the single-stranded DNA used in this experiment to form the double-ended hairpin structure is shown in SEQ ID NO:5.

[0076] The reaction system and reaction procedure used in this experiment are shown in Table 2-3:

[0077] Table 2: Exonuclease Activity Detection System

[0078] Double distilled water Add to 20μL 10×Taq buffer 2μL 2.5mM dNTP 2μL 100 μM hairpin structure DNA (SEQ ID NO:5) 1μL Taq DNA polymerase (1 U / uL) 1 / 2 / 3 / 4 / 5μL

[0079] Table 3: Procedure for detecting exonuclease activity:

[0080] Amplification program 55℃15s 99

[0081] By measuring the fluorescence intensity of the reaction at different concentrations of Taq DNA polymerase, a standard curve of the 5' to 3' exonuclease activity of wild-type Taq DNA polymerase can be plotted.

[0082] 2. Detection of polymerase activity of wild-type Taq-DNA polymerase

[0083] Design a single-stranded DNA that can form a single-ended hairpin structure, which can form a stable hairpin structure during extension at 72°C (e.g., ...). Figure 2 (As shown). The sequence of the single-stranded DNA forming the single-end hairpin structure is shown in SEQ ID NO:6.

[0084] Picogreen (purchased from Thermo Fisher Scientific, catalog number: P7589) is an extremely sensitive fluorescent nucleic acid dye that fluoresces only upon binding to double-stranded DNA, and the fluorescence is directly proportional to the concentration of double-stranded DNA. Adding Picogreen to the reaction system causes the fluorescence signal to gradually increase with the increase in DNA molecules as the primer extends, thus reporting the increase in DNA molecules in real time. Therefore, by detecting the intensity of the fluorescence signal, the concentration / quantity of DNA molecules can be determined, and consequently, the level of polymerase activity (e.g., ...). Figure 2(As shown).

[0085] The polymerase activity assay system and reaction procedure used in this experiment are shown in Table 4-5:

[0086] Table 4: Polymerase Activity System

[0087] Double distilled water Add to 20μL 10×Taq buffer 2μL 2.5mM dNTP 2μL 100 μM hairpin structure DNA (SEQ ID NO:6) 1μL Taq DNA polymerase (1 U / uL) 1 / 2 / 3 / 4 / 5μL 40X Picogreen 0.5uL

[0088] Table 5: Polymerase Activity Assay Procedure

[0089] Reaction Procedure 72℃15s 99

[0090] By measuring the fluorescence intensity of the reaction at different concentrations of Taq DNA polymerase, a standard curve of Taq DNA polymerase activity can be plotted.

[0091] The method for plotting the standard curve is as follows. In short, as described above, an enzyme activity assay reaction is performed using a specified amount of Taq DNA polymerase. The changes in fluorescence signal FU values ​​(ΔFU1, ΔFU2, ΔFU3, ΔFU4, ΔFU5) during the 1-minute reaction are measured and recorded under various polymerase dosage conditions. Subsequently, using ΔFU1 (1 U / µL of Taq DNA polymerase) as a base, the formula RFU = ΔFU5 is used to plot the standard curve. X / ΔFU1 was used to calculate RFU under various polymerase dosage conditions. Subsequently, a standard curve of enzyme activity was plotted with polymerase dosage on the x-axis and RFU on the y-axis.

[0092] Experimental results are as follows Figure 3 As shown. Figure 3 Standard curves for the polymerase activity and 5' to 3' exonuclease activity of wild-type Taq DNA polymerase, plotted using the two detection systems described above, are shown. The x-axis represents the concentration of wild-type Taq DNA polymerase, and the y-axis represents the RFU (reactive protein urinate) value. These curves can be used to quantitatively calculate the relative polymerase activity and relative 5' to 3' exonuclease activity of Taq DNA polymerase mutants.

[0093] Example 2: Detection of 5' to 3' exonuclease activity and polymerase activity of Taq-DNA polymerase mutants (E507K and Taq07)

[0094] The 5' to 3' exonuclease activities and polymerase activities of Taq-DNA polymerase mutants E507K and Taq07 were determined using the same methods as in Example 1. The relative enzyme activities of mutants E507K and Taq07 relative to wild-type Taq-DNA polymerase were calculated as follows. In short, 1 μL each of 1 U / μL E507K and Taq 07 polymerase were taken, reacted as described in Example 1, and the ΔFU values ​​were measured and recorded. Subsequently, the RFU values ​​of E507K and Taq 07 polymerase at this dosage were calculated as described in Example 1, and the relative enzyme activities of E507K and Taq 07 polymerase relative to wild-type Taq-DNA polymerase were determined based on the standard curve equation.

[0095] Experimental results are as follows Figure 4 As shown. Figure 4 A shows the relative polymerase activities of wild-type Taq DNA polymerase (WT), E507K, and Taq 07 (with WT polymerase activity as a reference); Figure 4 B shows the relative 5' to 3' exonuclease activities of WT, E507K, and Taq 07 (with WT's 5' to 3' exonuclease activity as a reference). The results indicate that Taq 07's polymerase activity is approximately 6 times that of WT and approximately 1.5 times that of E507K; and the exonuclease activity assay results show that Taq 07 has no 5' to 3' exonuclease activity.

[0096] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University; Xiamen Zhishan Biotechnology Co., Ltd. <120> A DNA polymerase mutant and its application <130> IDC200413 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of wild-type Taq DNA polymerase <400> 1 Met Ala Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 <210> 2 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of mutated Taq 07 polymer <400> 2 Met Ala Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Path Gly Arg Path Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Path Asp Asp Val Leu Path Ser Leu Path Lys Lys 115 120 125 Path Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Path Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Path Asp Tyr Arg Path Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Path To Arg Glu Lys To Leu Path His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Val Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Gly Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asn Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 <210> 3 <211> 2499 <212> DNA <213> Artificial Sequence <220> <223> A sequence encoding a mutated Taq 07 polymerase <400> 3 atggcggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg ggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccagag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaaggg cgaggaagct cctggaggag tggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acgtgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg ggaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctgggg 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc tagtagctc caatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tcaggcgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280. atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agaggcgga ggccgtggcc 2400. cggctggcca aggaggtcat ggaggggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtaa <210> 4 <211> 832 <212> PRT <213> Artificial Sequence <220> <223> Equivalent of Taq E507K wheelbase with smooth surface <400> 4 Met Ala Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Lys Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 <210> 5 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> Sequence of oligonucleotide probes forming double-ended hairpin structures <400> 5 caccgctggg cgcgatctgc cgcgcccagc ggtgacgtat aggtcctagc tacatgaacc 60 ccggcgccgt agatctgcct acggcgccgg gg 92 <210> 6 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> The sequence of oligonucleotides that form a single-ended hairpin structure <400> 6 cgcgcccagc ggtgacgtat aggtcctagc tacatgaacc ccggcgccgt agatctgcct 60 acggcgccgg gg 72

Claims

1. A mutant Taq DNA polymerase, characterized in that, The mutant Taq DNA polymerase, compared to the wild-type Taq DNA polymerase shown in SEQ ID NO: 1, undergoes the following mutations: L345V, E520G, and D578N.

2. The mutant Taq DNA polymerase of claim 1, wherein the polymerase activity is at least about 2, at least 3, at least 4, at least 5, or 6 times that of the wild-type Taq DNA polymerase; and / or, the exonuclease activity of the Taq DNA polymerase does not exceed about 10%, 5%, or 1% that of the wild-type Taq DNA polymerase.

3. The mutant Taq DNA polymerase of claim 1, wherein, The Taq DNA polymerase does not actually have 5' to 3' exonuclease activity.

4. The mutant Taq DNA polymerase of claim 1, wherein, The amino acid sequence of the mutated Taq DNA polymerase is shown in SEQ ID NO:

2.

5. A nucleic acid comprising a nucleotide sequence encoding a mutated Taq DNA polymerase as described in any one of claims 1-4.

6. The nucleic acid of claim 5, wherein, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

3.

7. A vector comprising the nucleic acid as described in claim 5 or 6.

8. The vector of claim 7, wherein, The vector is a plasmid, a granule, or a bacteriophage.

9. A host cell comprising the nucleic acid of claim 5 or 6 or the vector of claim 7 or 8.

10. A method of preparing the mutant Taq DNA polymerase of any one of claims 1-4, comprising: The mutant Taq DNA polymerase is expressed in a host cell, and then the mutant Taq DNA polymerase is recovered from the culture of the host cell.

11. Use of the mutated Taq DNA polymerase according to any one of claims 1-4, for: 1) To synthesize or amplify nucleic acids; or 2) Polymerase reaction activated by pyrophosphate hydrolysis (PAP); or 3) Analyze or determine the nucleotide sequence of nucleic acid molecules.

12. The use as described in claim 11, wherein, The pyrophosphate hydrolysis activated polymerase reaction (PAP) is used to detect rare mutations.

13. The use as described in claim 11, wherein, The nucleotide sequence analysis or determination process includes the following steps: incubating a primer molecule capable of hybridizing with the nucleic acid molecule, the nucleic acid molecule, and the mutated Taq DNA polymerase; and determining the nucleotide sequence of at least a portion of the nucleic acid molecule.

14. A kit comprising the mutated Taq DNA polymerase according to any one of claims 1-4.

15. The kit of claim 14, wherein, The kit can be used for nucleic acid synthesis or amplification, PAP or sequencing reactions.

16. The kit of claim 14, wherein, The kit also includes reagents selected from the following: reagents for PCR; reagents for PAP; reagents for sequencing reactions; or any combination thereof.

17. The kit of claim 16, wherein the kit comprises one or more of the following features: (i) The reagents used for PCR are selected from: buffers, dNTPs, and / or primers; (ii) The reagents used for PAP are selected from: buffer solutions, and / or pyrophosphate or similar substances; (iii) The reagents used for the sequencing reaction are selected from: buffers, dNTPs, primers, and / or, synthesis terminators.

18. A composition comprising the mutant Taq DNA polymerase of any one of claims 1-4, or the nucleic acid of claim 5 or 6, or the vector of claim 7 or 8, or the host cell of claim 9.

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