Taq DNA polymerase mutant as well as preparation method and application thereof

Abstract

The invention discloses a Taq DNA polymerase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The Taq DNA polymerase mutant is mutation on the basis of a wild type Taq DNA polymerase amino acid sequence as shown in SEQ ID NO.1, and comprises at least one of amino acid mutation L345E, L352D and S438M. The Taq DNA polymerase mutant has excellent tolerance to urea, and solves the dual technical problems that complex biological samples containing endogenous inhibitors such as urea can be efficiently amplified, and the Taq DNA polymerase mutant is compatible with difficult PCR reactions which take urea as an additive to optimize a high GC template and the like. Therefore, the mutant is a molecular biology tool enzyme which is wider in applicability and more stable and reliable in performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Taq DNA polymerase mutant, a preparation method and application thereof. BACKGROUND

[0002] Polymerase chain reaction (PCR) is the core technology of modern biotechnology, the key of which is to use heat-stable DNA polymerase to amplify specific DNA fragments exponentially in vitro. Such enzymes must be able to withstand repeated high-temperature cycles during PCR, and their catalytic efficiency and stability in complex reaction environments are key factors that determine the success or failure of applications such as scientific research, clinical diagnosis, and forensic science.

[0003] Taq DNA polymerase derived from thermophilic bacteria is the most widely used heat-stable polymerase, and is widely used in scientific research, clinical diagnosis, and forensic science. However, PCR reactions are often affected by inhibitors in samples, and urea is one of the common ones, which may come from clinical samples, plant tissues, or food degradation products. On the other hand, urea itself is also an effective PCR reaction enhancer, which is often added actively to assist the amplification of high GC content templates and improve reaction specificity.

[0004] Although urea has application value, conventional Taq DNA polymerase is highly sensitive to it. As a protein denaturant, urea can significantly inhibit the activity of Taq enzyme through multiple pathways. First, it destroys the internal structural network of the enzyme by forming hydrogen bonds with the enzyme protein, weakening the stability of its spatial conformation; second, it competes with DNA for binding to the active center of the enzyme, thereby interfering with the effective binding of the substrate; third, it can bind to magnesium ions necessary for catalysis, thereby inhibiting the catalytic activity of the enzyme.

[0005] Therefore, there is a clear technical contradiction in the prior art: whether as an unavoidable contaminant in samples or as an active additive required for reaction optimization, the presence of urea has severely inhibited the activity of standard Taq enzyme, resulting in a significant decrease in amplification efficiency and detection sensitivity, and even reaction failure. This sensitivity to urea makes Taq enzyme perform poorly when dealing with complex biological samples or performing high-performance PCR optimization, greatly limiting the application range and reliability of PCR technology, and constituting a technical bottleneck that needs to be solved in this field.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The application aims to provide a Taq DNA polymerase mutant, a preparation method and application thereof. The Taq DNA polymerase mutant has excellent urea tolerance, can efficiently amplify complex samples containing endogenous inhibitors, and is compatible with a PCR optimization system with urea as an additive, and is a more widely applicable and more robust tool enzyme.

[0008] In order to achieve the above-mentioned purpose of the application, the following technical solutions are adopted: In a first aspect, the application provides a Taq DNA polymerase mutant, which is based on a mutation of a wild-type Taq DNA polymerase amino acid sequence as shown in SEQ ID NO. 1, and contains at least one selected from amino acid mutations L345E, L352D and S438M.

[0009] In an optional embodiment, the amino acid sequence of the amino acid mutation L345E is shown in SEQ ID NO. 2; and / or, the amino acid sequence of the amino acid mutation L352D is shown in SEQ ID NO. 3; and / or, the amino acid sequence of the amino acid mutation S438M is shown in SEQ ID NO. 4.

[0010] In a second aspect, the application provides a polynucleotide molecule encoding the Taq DNA polymerase mutant as described in the preceding embodiments.

[0011] In a third aspect, the application provides a recombinant expression vector containing the polynucleotide molecule as described in the preceding embodiments.

[0012] In a fourth aspect, the application provides a host cell containing the recombinant expression vector as described in the preceding embodiments; or, a chromosome integrated with the polynucleotide molecule as described in the preceding embodiments.

[0013] In a fifth aspect, the application provides a kit comprising the Taq DNA polymerase mutant as described in the preceding embodiments.

[0014] In a sixth aspect, the application provides a preparation method of the Taq DNA polymerase mutant as described in the preceding embodiments, comprising: constructing a recombinant expression vector containing a polynucleotide molecule encoding the Taq DNA polymerase mutant; transforming the recombinant expression vector into a host cell; culturing the transformed host cell and inducing expression of the Taq DNA polymerase mutant; purifying the Taq DNA polymerase mutant from the culture.

[0015] In a seventh aspect, the present application provides a method for preparing the Taq DNA polymerase mutant according to the preceding embodiments, wherein the host cell is E. coli; and / or, The step of constructing the recombinant expression vector is achieved by performing rolling circle PCR using a plasmid containing the wild-type Taq DNA polymerase gene as a template; and / or, The induced expression is achieved by adding IPTG in the culture medium; and / or, The step of isolating and purifying includes isolating the Taq DNA polymerase mutant by affinity chromatography.

[0016] In an eighth aspect, the present application provides a method for amplifying target DNA, comprising: using the Taq DNA polymerase mutant according to the preceding embodiments in a polymerase chain reaction; Preferably, the polymerase chain reaction is performed in a reaction system containing urea. Preferably, the method is used for amplifying high-GC-content templates, or for DNA amplification of clinical, food or plant samples containing urea.

[0017] In a ninth aspect, the present application provides the use of the Taq DNA polymerase mutant according to the preceding embodiments for amplifying target DNA in a polymerase chain reaction containing urea.

[0018] The present application provides a Taq DNA polymerase mutant, as well as a method for preparing and using the same. Compared with the prior art, the Taq DNA polymerase mutant provided by the present application has mutations of L345E, L352D or S438M, and exhibits the following remarkable advantages compared with the wild-type Taq DNA polymerase: Firstly, the most core advantage of the Taq DNA polymerase mutant is its significant tolerance to urea inhibition. In a reaction environment containing urea, its ability to amplify target DNA fragments is much higher than that of the wild-type enzyme. Experimental data show that the amount of amplification product can reach 2.62 to 5.04 times that of the wild-type. This significant improvement in performance means that the enzyme can still maintain high catalytic activity in the presence of urea inhibitors.

[0019] Secondly, this resistance to urea makes the enzyme more reliable and efficient in dealing with complex biological samples. In applications such as clinical diagnosis, food detection or plant gene cloning, samples often contain endogenous urea or similar metabolites, which can inhibit the activity of conventional Taq enzyme, leading to amplification failure or low efficiency. However, the mutant enzyme can work effectively in these "crude" samples containing inhibitors, thereby improving the success rate and sensitivity of detection and simplifying the sample pretreatment steps.

[0020] Furthermore, the characteristics of the mutant enzyme solve a common contradiction in PCR optimization. Urea itself is a useful PCR reaction additive that can assist in amplifying difficult templates with high GC content by lowering the DNA melting temperature and reducing non-specific binding. However, the sensitivity of wild-type Taq enzyme to urea limits the application of this strategy. The mutant enzyme, due to its resistance to urea, allows the addition of an effective concentration of urea in the PCR reaction to optimize the reaction conditions, thereby successfully amplifying previously difficult-to-handle DNA templates, expanding the application range of PCR technology, and making the construction of reaction systems more convenient.

[0021] In summary, the Taq DNA polymerase mutant, through specific amino acid substitution, has obtained the powerful ability to maintain activity in complex chemical environments. This enhanced robustness not only makes it perform better in the face of inhibitor-containing samples, but also enables it to be compatible with the optimization system using urea as an enhancer, ultimately making it a more widely applicable and reliable molecular biology tool enzyme. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0023] Figure 1 Electrophoresis results of Taq variants and wild-type purified crude enzyme in Example 2 of the present application; Figure 2 Amplification performance difference results of Taq variants and wild-type in urea-free / urea-containing systems in Example 3 of the present application. DETAILED DESCRIPTION

[0024] The embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not noted by the manufacturer, which are all conventional products that can be obtained by purchase.

[0025] In the embodiments of the present application, a Taq DNA polymerase mutant is provided, which is based on the mutation of the wild-type Taq DNA polymerase amino acid sequence as shown in SEQ ID NO. 1, and contains at least one selected from the amino acid mutations L345E, L352D and S438M.

[0026] The above-mentioned Taq DNA polymerase is a protein, and its function is to synthesize DNA. This particular substance is a "mutant" or "variant" obtained by changing the amino acid sequence of the naturally occurring "wild-type" Taq DNA polymerase with the sequence of SEQ ID NO. 1. Among the parts included, the basic component is a protein, which is composed of a polypeptide chain, and the polypeptide chain is composed of a plurality of amino acids connected in a specific order.

[0027] In the structural framework, the vast majority of its amino acid sequence is exactly the same as the wild-type Taq DNA polymerase shown in SEQ ID NO. 1.

[0028] The core component is the key difference from the wild-type enzyme, which contains at least one specific amino acid substitution. These substitutions are: (1) At position 345 of the sequence, the original leucine (L) is replaced by glutamic acid (E).

[0029] (2) At position 352 of the sequence, the original leucine (L) is replaced by aspartic acid (D).

[0030] (3) At position 438 of the sequence, the original serine (S) is replaced by methionine (M).

[0031] It should be noted that a specific product can only contain one of the substitutions, or can contain a combination of two or all three substitutions.

[0032] The most remarkable advantage of this substance is its strong tolerance to urea inhibition. In reaction systems containing urea, its DNA amplification performance is significantly better than that of the unmodified wild-type enzyme, and the product yield can reach 2.62 to 5.04 times that of the wild type. This core advantage brings two practical application effects: first, it can maintain high activity when dealing with complex biological samples (such as clinical, food, and plant samples) containing endogenous inhibitors (such as urea), thereby improving the success rate and reliability of DNA amplification. Second, the tolerance of this substance also allows the active addition of urea as an enhancer in PCR reactions to help amplify difficult DNA templates with high GC content or improve reaction specificity, which provides convenience for optimizing complex PCR reactions and broadens their application range.

[0033] In some embodiments, the amino acid sequence of the amino acid mutation L345E is shown in SEQ ID NO. 2.

[0034] In some embodiments, the amino acid sequence of the amino acid mutation L352D is shown in SEQ ID NO. 3.

[0035] In some embodiments, the amino acid sequence of the amino acid mutation S438M is shown in SEQ ID NO. 4.

[0036] In the embodiments of the present application, a polynucleotide molecule encoding the Taq DNA polymerase mutant as described in the preceding embodiments is provided.

[0037] In the embodiments of the present application, a recombinant expression vector comprising the polynucleotide molecule as described in the preceding embodiments is provided.

[0038] In the embodiments of the present application, a host cell containing the recombinant expression vector as described in the preceding embodiments, or having the polynucleotide molecule as described in the preceding embodiments chromosomally integrated, is provided.

[0039] In the embodiments of the present application, a kit comprising the Taq DNA polymerase mutant as described in the preceding embodiments is provided.

[0040] The product combination provided in the embodiments of the present application is a product combination, i.e., a "kit", in which a plurality of chemical reagents for performing a polymerase chain reaction (PCR) are pre-packaged together. The DNA polymerase it contains is the aforementioned Taq DNA polymerase mutant with L345E, L352D, or S438M mutations, which is tolerant to urea, rather than the standard wild-type Taq enzyme.

[0041] A typical kit will contain the main components required for performing a PCR experiment for the convenience of use. According to the content, the kit must include: (1) Core component: Taq DNA polymerase mutant as described above.

[0042] (2) In order to constitute a functionally complete PCR kit, it usually also contains other necessary components, for example, which can include but are not limited to: A reaction buffer (PCR Buffer): Provide a chemical environment suitable for enzyme activity (such as pH, ion concentration, etc.). The buffer usually contains potassium chloride (KCl), tris-hydroxymethyl aminomethane hydrochloride (Tris-HCl) and magnesium chloride (MgCl2) which is essential for enzyme activity.

[0043] B Deoxyribonucleotide triphosphate (dNTPs): A mixture of dATP, dGTP, dCTP, dTTP, which is the raw material for DNA synthesis.

[0044] C Instructions for use: Guide users on how to correctly use the kit for experiments.

[0045] In the embodiments of the present application, a preparation method of the Taq DNA polymerase mutant as described in the foregoing embodiments is provided, comprising: Step S1, constructing a recombinant expression vector containing a polynucleotide molecule encoding the Taq DNA polymerase mutant.

[0046] The purpose of this step is to create a circular DNA molecule (plasmid) containing the gene encoding the target Taq enzyme mutant for subsequent expression in host cells.

[0047] This process can use a pET30a plasmid containing the wild-type Taq enzyme gene as a template. First, according to the wild-type Taq nucleic acid sequence (SEQ ID NO. 5), specific point mutation primers are designed for the three sites of L345E, L352D and S438M. Then, using rolling circle PCR technology, amplification is carried out in a reaction system containing the template plasmid, primers, high-fidelity DNA polymerase, etc. After amplification, the original, unmutated methylated template plasmid is digested using DpnI endonuclease, while the newly synthesized plasmid containing the mutation is retained.

[0048] Step S2, transforming the recombinant expression vector into host cells.

[0049] The purpose of this step is to introduce the plasmid carrying the mutant gene constructed in the previous step into host cells that can produce the protein.

[0050] The process can choose E. coli BL21 DE3 as the competent host cell. The product after Dpnl digestion is added to the competent cell and placed on ice for 30 minutes. Then, the cell membrane is made to produce transient permeability by 42°C water bath heat shock for 90 seconds, so as to introduce the plasmid into the cell. After heat shock, LB medium is added, and the cell is recovered at 37°C for 50 minutes. Finally, the recovered bacterial solution is coated on an LB solid plate containing kanamycin (Kan) resistance for screening. Only bacteria successfully introducing the plasmid can obtain resistance and grow, so a single colony can be formed on the plate. The single colony is picked for sequencing to finally confirm that the engineering strain containing the correct mutant sequence is obtained.

[0051] Step S3, the transformed host cell is cultured, and the expression of the Taq DNA polymerase mutant is induced.

[0052] The purpose of this step is to culture the engineering strain in large quantities and induce it to produce the target Taq enzyme mutant protein. The process can be divided into two stages of culture: First, the correct strain is inoculated into at least an amount of LB medium for overnight culture of the first-stage seed solution. Then, the first-stage seed solution is transferred to a larger volume of secondary culture flask for further culture until the bacterial solution concentration reaches about OD600 0.6. At this time, IPTG with a final concentration of 1 mM is added to the culture medium as an inducer to start the expression of the Taq enzyme mutant gene. After adding IPTG, the culture temperature is reduced to 20°C, and the overnight culture is continued to allow the cell to synthesize and accumulate the target protein in large quantities.

[0053] Step S4, the Taq DNA polymerase mutant is separated and purified from the culture.

[0054] The purpose of this step is to extract the target protein from a large number of bacterial bodies and purify it. First, the bacterial bodies after induction culture can be collected by centrifugation, and resuspended by adding a cell lysis solution containing lysozyme. The cell wall is lysed by cycles of -80°C freezing and 37°C treatment to release the intracellular protein. Again, centrifugation is used to remove cell debris, and the clear supernatant containing the target protein is collected. Then, affinity chromatography is used for purification, and the supernatant is passed through a nickel column to allow the target protein with a special tag to be adsorbed on the column. After washing with a washing solution to remove impurities, the target protein is eluted from the column with an elution solution containing a high concentration of imidazole. Finally, the collected eluate is dialyzed overnight to remove imidazole and replace it with a suitable storage buffer, and finally the purified crude enzyme is obtained. The purity and molecular weight of the obtained enzyme can be verified by SDS-PAGE electrophoresis.

[0055] In some embodiments, the host cell is E. coli.

[0056] The above-mentioned E. coli is used as a host cell. In a specific implementation, the BL21 DE3 competent cell of E. coli can be selected.

[0057] In some embodiments, the step of constructing the recombinant expression vector is completed by performing rolling circle PCR using a plasmid containing a wild-type Taq DNA polymerase gene as a template.

[0058] The above-mentioned rolling circle PCR can be performed using a plasmid (pET30a plasmid) containing a wild-type Taq DNA polymerase gene as an amplification template, and using primers specially designed for introducing specific point mutations, under specific PCR systems and programs. This method can directly introduce point mutations on the original plasmid, and is efficient.

[0059] In some embodiments, the induced expression is achieved by adding IPTG to the culture medium.

[0060] This step can be achieved by adding IPTG to the culture medium. After the strain is cultured to an appropriate concentration (OD600 is about 0.6), IPTG is added to the culture medium to a final concentration of 1 mM, thereby starting the transcription and translation of the target gene, and making the host cell start producing Taq enzyme mutants in large quantities.

[0061] In some embodiments, the step of isolating and purifying includes isolating the Taq DNA polymerase mutant by affinity chromatography.

[0062] This method refines the technical means of protein purification, which includes isolating the target product by affinity chromatography. In a specific example, affinity chromatography for a tag protein can be used, that is, a method of passing through a nickel column. After cell disruption, the supernatant flows through the nickel column, and the Taq enzyme mutant with a specific tag is specifically adsorbed on the column, while most of the impurities flow through. Subsequently, by elution, a relatively pure target protein can be obtained.

[0063] In the embodiments of the present application, a method for amplifying a target DNA is provided, which comprises: using the Taq DNA polymerase mutant as described in the foregoing embodiments in a polymerase chain reaction.

[0064] The core of the above-mentioned method is to use a Taq DNA polymerase mutant containing at least one L345E, L352D or S438M mutation to catalyze the synthesis of DNA in a polymerase chain reaction (PCR). In a specific experimental operation, the mutant enzyme can be added to a reaction system containing 10x PCR buffer, dNTPs, primers and DNA template, and amplified by the program set by the PCR instrument.

[0065] In some embodiments, the polymerase chain reaction is performed in a reaction system containing urea.

[0066] One preferred embodiment of the method is to include urea in the PCR reaction system. This is because the most significant advantage of the mutant Taq enzyme is its tolerance to urea. Under conditions containing urea, the amplification performance of the mutant enzyme is much better than that of the wild-type enzyme, and the brightness of the amplification product can be 2.62 to 5.04 times that of the wild-type. Therefore, the use of the enzyme in the presence of urea best reflects its technical advantages.

[0067] In some embodiments, the method is used to amplify high GC content templates, or to perform DNA amplification on clinical, food or plant samples containing urea.

[0068] Another preferred embodiment of the method is its application to specific amplification scenarios.

[0069] In terms of amplifying high GC content templates, the method is particularly suitable for amplifying high GC content DNA templates. It is mentioned in the background art that urea is often used as an enhancer to treat such difficult-to-unwind templates. Since the mutant enzyme is tolerant to urea, it can work synergistically with urea to effectively solve the problem of difficult amplification of high GC templates.

[0070] In terms of processing complex biological samples, the method is also particularly suitable for directly amplifying DNA from complex biological samples containing urea or similar inhibitors. These samples include clinical samples, food samples or plant samples, which often contain substances that inhibit PCR reactions. The use of the tolerant mutant enzyme can overcome these inhibitory effects, thereby improving the success rate and reliability of detection.

[0071] In the examples of the present application, a mutant Taq DNA polymerase as described in the preceding embodiments is provided for use in amplifying target DNA in a polymerase chain reaction containing urea.

[0072] The present application is further illustrated by the following specific examples, but it should be understood that these examples are merely intended to illustrate in more detail and should not be construed as limiting the present application in any form.

[0073] Example 1: Construction of Taq variants In this example, Taq variants were constructed by rolling circle PCR using a pET30a plasmid containing the wild-type Taq sequence as a template.

[0074] Experimental methods: (1) Based on the wild-type Taq nucleic acid sequence (SEQ ID NO.5), rolling circle amplification primers for point mutations L345E, L352D and S438M were designed. See the primer synthesis summary table 1. The rolling circle PCR amplification system was prepared according to Table 2 and the program in Table 3 was used to amplify the target fragment. After digesting the template with DpnI, the cells were transformed into Escherichia coli competent cells BL21 DE3.

[0075] Table 1. Summary of primers used for Taq variant construction

[0076] Table 2. Rolling circle PCR amplification system for Taq variants

[0077] Table 3. Taq variant construction rolling circle PCR amplification procedure

[0078] (2) Take 100 µL of BL21 DE3 competent cells from the -80 ℃ freezer and thaw them on ice. Add 10 μL of the digested product to each competent cell, gently pipette to mix, and let stand on ice for 30 min. Then heat shock in a 42 ℃ water bath for 90 s, immediately cool in an ice-water bath, and add 750 μL of LB liquid medium after 3 min. Recover at 37 ℃ in a shaker for 50 min. After recovery, centrifuge at 4000 rpm for 2 min. Remove the supernatant, resuspend the bacterial cells in the remaining 100 μL of liquid, and spread evenly on LB Kan solid plates. Incubate upside down at 37 ℃ for about 12 h. Sequencing of single colonies on the plates confirms the correct sequence and obtains the Taq variant.

[0079] Example 2: Preparation of Taq variant and wild-type crude enzyme In this embodiment, Taq variant and wild-type crude enzymes were prepared.

[0080] Experimental methods: 1. Taq variant and wild-type culture induction: (1) Primary seed culture: Taq variant and wild type were inoculated into 15 mL centrifuge tubes (2 mL LB medium, 50 μg / mL Kan) and cultured overnight at 37 °C and 250 rpm.

[0081] (2) Secondary induction: Take 1 mL of culture medium from the primary seed culture and transfer it to a secondary 250 mL shake flask containing 50 μL LB medium, 50 μg / mL Kan, and culture at 37 ℃ and 250 rpm until the OD600 is about 0.6. Add a certain amount of IPTG (final concentration of 1 mM) to induce enzyme expression, and induce overnight at 20 ℃ and 250 rpm.

[0082] 2. Cell disruption: The well-cultured secondary bacteria solution was centrifuged to collect the bacterial cells, 5 mL of cell disruption solution (50 mM Tris pH 7.5, 0.3% lysozyme) was added, mixed well, and then placed at -80 °C for 30 min and treated at 37 °C for 30 min. The mixture was centrifuged at 12000 rpm for 20 min, and the supernatant was collected.

[0083] 3. Preparation of crude enzyme: The supernatant was passed through a nickel column to adsorb the target protein, and then 3 mL of washing solution (20 mM Tris, 20 mM NaCl, 20 mM imidazole, pH 7.5) was used to wash three times, and then 0.5 mL of elution solution (20 mM Tris, 20 mM NaCl, 500 mM imidazole, pH 7.5) was used for elution. The obtained eluate was transferred to a 30 kD dialysis bag and placed in dialysis solution. The dialysis solution was replaced every 2-3 hours and dialyzed overnight. The crude enzyme was collected. The expression of the crude enzyme was verified by SDS-PAGE, and the protein concentration was quantified according to the BSA standard, as shown in Figure 1 .

[0084] Example 3 In this example, Experimental method: 1. Urease inhibition test of Taq variants and wild type (1) Preparation of amplification system: Table 4. Amplification system without and with urea for Taq variants and wild type

[0085] Note: 10x PCR Buffer buffer (100 mM Tris-HCl, 500 mM KCl, 30 mM MgCl2, pH 8.3 (25 °C)).

[0086] The normal amplification system was prepared in the PCR tube according to the above table, and after preparation, it was vortexed for 10 s in a vortex mixer and centrifuged for a few seconds in a microcentrifuge.

[0087] 2. Normal amplification reaction Table 5. Amplification program of Taq variants and wild type crude enzyme

[0088] The PCR tube was placed in a PCR instrument to execute the above program; 3. Gel migration experiment of normal amplification reaction product After completion, 5 μΐ of the reaction product was added to 1 μΐ of 6x loading buffer, mixed, and loaded into an agarose gel electrophoresis well. The gel was run using 1x TBE running buffer at 150 V for 12 min. The products were photographed using ultraviolet conditions on a ChampGel 5000 imaging system (Suzhou Nanchen) and the brightness (gray value analysis) of the Taq variant and wild-type amplification bands was analyzed using ImageJ.

[0089] 4. Experimental results: The experimental results are shown in Table 6. Figure 2 The gray scale analysis results are shown in Table 6. Figure 2

[0090] Table 6. Statistics Figure 2 Taq variant and wild-type amplification ability in the presence / absence of urea

[0091] Figure 2 Taq variant and wild-type amplification ability in the presence / absence of urea: lanes 1, 2, 3, and 4 are the amplification ability of the Taq variant and wild-type crude enzyme in the absence of urea; lanes 5, 6, 7, and 8 are the differences in amplification ability of the Taq variant and wild-type crude enzyme in the presence of urea.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacements to some or all of the technical features; and these modifications or replacements do not cause the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.​

Claims

1. A mutant of Taq DNA polymerase, characterized in that, The Taq DNA polymerase mutant is based on mutations of wild-type Taq DNA polymerase amino acid sequence as shown in SEQ ID NO. 1, and comprises at least one selected from amino acid mutations L345E, L352D and S438M.

2. The mutant Taq DNA polymerase according to claim 1, wherein The amino acid sequence of the amino acid mutation L345E is shown in SEQ ID NO. 2; and / or, The amino acid sequence of the amino acid mutation L352D is shown in SEQ ID NO. 3; and / or, The amino acid sequence of the amino acid mutation S438M is shown in SEQ ID NO.

4.

3. A polynucleotide molecule, characterized in that, The recombinant expression vector comprises the polynucleotide molecule as claimed in claim 3.

4. A recombinant expression vector, characterized in that, The host cell contains the recombinant expression vector as claimed in claim 4; or, the polynucleotide molecule as claimed in claim 3 is chromosomally integrated.

5. A host cell, characterized in that, The kit comprises the Taq DNA polymerase mutant as claimed in claim 1 or 2.

6. A kit characterized in that, Comprising:

7. A method for preparing the mutant Taq DNA polymerase according to claim 1 or 2, characterized by, constructing a recombinant expression vector comprising a polynucleotide molecule encoding the Taq DNA polymerase mutant; transforming the recombinant expression vector into a host cell; culturing the transformed host cell, and inducing expression of the Taq DNA polymerase mutant; isolating and purifying the Taq DNA polymerase mutant from the culture. The host cell is Escherichia coli; and / or, 8. The method for preparing Taq DNA polymerase mutant according to claim 7, wherein the mutant is a mutant of the amino acid sequence of SEQ ID NO:

1. The step of constructing the recombinant expression vector is accomplished by performing rolling circle PCR with a plasmid containing the wild-type Taq DNA polymerase gene as a template; and / or, The step of inducing expression is achieved by adding IPTG in the culture medium; and / or, The step of isolating and purifying comprises isolating the Taq DNA polymerase mutant by affinity chromatography. Comprising:

9. A method for amplifying a target DNA, characterized by, using the Taq DNA polymerase mutant as claimed in claim 1 or 2 in a polymerase chain reaction; Preferably, the polymerase chain reaction is performed in a reaction system containing urea; Preferably, the method is used for amplifying high GC content templates, or for DNA amplification of clinical, food or plant samples containing urea.

10. Use of the Taq DNA polymerase mutant as claimed in claim 1 or 2 for amplifying target DNA in a polymerase chain reaction containing urea. ​

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