CN111826366B

CN111826366B - Direct-amplification type hot-start DNA polymerase and preparation method and application thereof

Info

Publication number: CN111826366B
Application number: CN202010503288.XA
Authority: CN
Inventors: 胡松青; 黄丽芳; 苑歆; 刘光毅; 何贤蓉; 侯轶; 樊壬水
Original assignee: Guangzhou Yingzan Biotechnology Co ltd
Current assignee: Guangzhou Yingzan Biotechnology Co ltd
Priority date: 2020-06-05
Filing date: 2020-06-05
Publication date: 2022-08-02
Anticipated expiration: 2040-06-05
Also published as: CN111826366A

Abstract

The invention discloses a direct-amplification hot-start DNA polymerase, a preparation method and application thereof. The direct amplification type hot start DNA polymerase disclosed by the invention is prepared by carrying out deletion, site-specific mutagenesis and fusion of nonspecific double-stranded DNA binding protein domain biological mutagenesis on wild type Taq DNA polymerase and carrying out chemical modification. The direct amplification type hot start DNA polymerase can greatly improve the stability of the DNA polymerase, can tolerate high-concentration fluorescent dye and various inhibitors, can be applied to direct PCR or qPCR detection of various sources and complex samples, and enables PCR related reaction and detection to be more convenient. The invention also discloses a qPCR reaction kit which has high detection specificity and strong fluorescent signal, is suitable for rapid quantitative detection of low-content crude extracted nucleic acid, and can be applied to food doping, microbial detection and transgenic plant screening.

Description

Direct-amplification type hot-start DNA polymerase and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to direct-amplification hot-start DNA polymerase and a preparation method and application thereof.

Background

The Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qPCR) technology is developed on the basis of Polymerase Chain Reaction (PCR), makes up for the defect that the common PCR can not be monitored and quantified in Real time, and becomes an important tool for the current gene research. The SYBR Green I dye method qPCR technology is widely applied to the fields of medical research, pathogenic microorganism detection, food safety detection and the like due to the advantages of low cost, good repeatability, strong applicability and the like.

Sensitivity and accuracy are always the development requirements and targets of the detection field, and the activity of Taq DNA polymerase is interfered by a sample or a residual qPCR inhibitor in the sample extraction process when the SYBR Green I dye method qPCR technology is applied to nucleic acid detection. For example, polysaccharides in plant nucleic acid samples, immunoglobulins in blood nucleic acid samples, humic acid in soil nucleic acid samples and the like can inhibit Taq DNA polymerase activity, thereby affecting specificity, fluorescent signals and sensitivity of qPCR detection and causing unreliable and inaccurate results.

Taq DNA polymerase is used as a core component of qPCR reaction, has super heat resistance and polymerase activity, and the performance of the Taq DNA polymerase directly determines the speed and specificity of qPCR detection and the reliability of a result. The hot start DNA polymerase can block the activity of the DNA polymerase before PCR reaction, so that the activity is released only when the PCR reaction starts, the non-specific amplification and the formation of primer dimerization can be greatly reduced, and the hot start DNA polymerase is a main component of the current qPCR reagent. In recent years, a variety of inhibitor-resistant Taq DNA polymerases have emerged that can directly perform PCR amplification on a crude sample. However, the current direct amplification Taq DNA polymerase has a small amount of crude sample, easily causes false negative results (e.g., detection of a sample at the initial stage of viral or bacterial infection) when the amount of a target to be detected in the sample is low, and is only suitable for general PCR. The Taq DNA polymerase used for the crude direct qPCR detection needs stronger inhibitor resistance so as to ensure the specificity of the qPCR detection and the reliability of the result in the presence of high-concentration inhibitors and high-concentration dyes. Therefore, there is a need to develop a high-performance hot-start DNA polymerase for qPCR.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing a direct-amplification type hot-start DNA polymerase.

Another object of the present invention is to provide a method for preparing the direct-amplification-type hot-start DNA polymerase.

It is still another object of the present invention to provide use of the direct-amplification-type hot-start DNA polymerase.

The purpose of the invention is realized by the following technical scheme:

a direct-amplification hot-start DNA polymerase has an amino acid sequence shown in SEQ ID NO. 1.

Compared with the amino acid sequence of wild type TaqDNA polymerase with NCBI accession number P19821.1, the direct amplification type hot start DNA polymerase has the following characteristics: 1-289 site amino acid deletion, 626 site glutamic acid mutation to arginine, 707 site isoleucine mutation to leucine, 708 site glutamic acid mutation to arginine, and N-terminal fusion of double-stranded DNA binding protein.

The double-stranded binding protein is preferably Sso7d with an amino acid sequence shown as SEQ ID NO. 2. The double-stranded binding protein can significantly improve the affinity of the DNA polymerase for the template DNA.

A DNA molecule encoding the direct-amplification hot-start DNA polymerase.

The nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 3. The sequence is obtained by codon optimization according to the characteristics of an escherichia coli expression system, and the expression efficiency of a heterologous gene in host bacteria can be obviously improved.

A recombinant expression vector is obtained by cloning a DNA molecule encoding the direct-amplification hot-start DNA polymerase into an expression vector.

The expression vector is preferably a prokaryotic expression vector; more preferably a pET series vector; most preferably pET-28 a.

A recombinant engineering cell strain is obtained by transforming the recombinant expression vector into engineering cells.

The engineering cell is preferably an Escherichia coli cell; more preferably BL21(DE3) cells. The recombinant engineering cell strain can express the recombinant expression vector quickly and solubly.

The direct-amplification hot-start DNA polymerase can be obtained by a chemical synthesis method or prepared by induced expression and purification of recombinant engineering cell strains. From the viewpoint of cost, it is preferably produced by inducing expression of a recombinant engineered cell line and purifying it; the method specifically comprises the following steps:

inoculating the recombinant engineering cell strain into an LB culture medium containing antibiotics, and culturing at 35-38 ℃ until the OD of a bacterial liquid ₆₀₀ And (3) reaching 0.7-0.9, inducing cells to express proteins by IPTG, and separating and purifying to obtain the direct amplification type hot start DNA polymerase.

The antibiotic is preferably kanamycin.

The amount of kanamycin is preferably calculated as 50. mu.g/mL of LB medium.

The usage amount of the IPTG is preferably calculated according to the final concentration of 0.1-0.5 mmol/L.

The inducing condition is preferably 35-38 ℃ and 2-4 h; more preferably, the temperature is 37 ℃ and the time is 3 hours.

The separation and purification method is preferably to perform nickel ion affinity chromatography (Ni-NTA chromatography) and then perform anion exchange column chromatography.

Preferably, the formula of the binding Buffer (Buffer a) used for the nickel ion affinity chromatography is as follows: 50mmol/L NaCl, 50mmol/L Tris-HCl, pH 8.0; the formulation of the elution Buffer (Buffer B) used was: 500mmol/L imidazole, 50mmol/L Tris-HCl, 50mmol/L NaCl, pH 8.0.

Preferably, the formulation of the binding Buffer (Buffer C) used for anion exchange chromatography is: 50mmol/L Tris-HCl, 100mmol/L NaCl, pH 8.0; the formulation of the elution Buffer (Buffer D) used was: 50mmol/L Tris-HCl, 1mol/L NaCl, pH 8.0.

Preferably, an anhydride compound is specifically bound to a lysine side chain amino group at the polymerase active site of the direct-amplification hot-start DNA polymerase, so that the polymerase activity can be reversibly blocked.

The anhydride compound is preferably cis-aconitic anhydride or citral estolide; more preferably citric anhydride. The anhydride modified polymerase greatly reduces the possibility of nonspecific product amplification and primer dimer formation in the PCR reaction process.

The preparation method of the direct amplification type hot start DNA polymerase comprises the following steps:

(1) dialyzing the direct-amplification hot-start DNA polymerase into a Tris-HCl buffer solution;

(2) adding an anhydride compound, uniformly mixing, and incubating;

(3) dialyzing the incubated enzyme solution into a storage buffer solution to obtain the stable direct-amplification hot-start DNA polymerase. The hot start DNA polymerase prepared by the method has excellent hot start performance, and no polymerase activity is released after incubation for 10min at 50 ℃; the activity can be released after the heat shock at 95 ℃ for 5-10 min.

The preferable concentration of the Tris-HCl buffer solution is 10-30 mmol/L, pH-9-10 Tris-HCl buffer solution.

The proportion of the direct amplification type hot start DNA polymerase to the anhydride compound is preferably 1: 50-1: 100 in a molar ratio.

The incubation condition is preferably 40-50 ℃ for 2-5 h.

The formula of the storage buffer is preferably as follows: 20mmol/L Tris-HCl, 100mmol/L KCl, 0.1mmol/L EDTA, 50% Glycerol (Glycerol), 1mmol/LDTT, 0.5% Tween-20, 0.5%

CA-630，pH8.0。

A PCR reaction buffer comprising the following components: 20 to 100mmol/L Tris-HCl, 1 to 3mmol/L MgCl ₂ 、5～20mmol/L(NH ₄ ) ₂ SO ₄ 20-80 mmol/L KCl, 0.1-0.4 mg/L BSA, 0.05-0.1% Tween-20, 1-6% DMSO, and the pH value is 8-10; the PCR reaction buffer solution is matched with the direct amplification type hot start DNA polymerase for use.

The PCR reaction buffer solution also comprises at least one of trehalose, ethyl phenyl polyethylene glycol (NP-40) and L-carnitine.

The concentration of the trehalose in the system is preferably 0.1-0.4 mol/L; more preferably 0.3 mol/L.

The concentration of the ethyl phenyl polyethylene glycol in the system is preferably 0-0.4%; more preferably 0.4%.

The concentration of the L-carnitine in the system is preferably 0.1-0.3 mol/L; more preferably 0.2 mol/L.

The application of the PCR reaction buffer solution in DNA sample amplification.

A qPCR reaction kit comprises the PCR reaction buffer solution, SYBR Green I, at least one of water for PCR and dNTPs, and the direct-amplification hot-start DNA polymerase.

The concentration of the SYBR Green I in the system is 0.2-10 ng/mu L.

The concentration of the direct amplification type hot start DNA polymerase in the system is preferably 0.02-0.05U/mu L.

The concentration of the dNTPs in the system is preferably 100-300 mu mol/L.

The qPCR reaction kit is applied to DNA sample amplification.

The DNA sample is not limited in source, and may contain at least one of humic acid, polysaccharide, immunoglobulin, casein and other inhibitors. For example, a plant nucleic acid sample containing polysaccharides, a blood nucleic acid sample containing immunoglobulins, a milk nucleic acid sample containing casein, a soil nucleic acid sample containing humic acid, and the like.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the direct-amplification hot-start DNA polymerase has good stability, and can tolerate high-concentration fluorescent dye and various high-concentration inhibitors in qPCR (quantitative polymerase chain reaction) detection, such as 10 ng/muL SYBR Green I, 1.6 mug/mL humic acid, 0.32mg/mL astragalus polysaccharide or 600 mug/mL IgG.

(2) The hot start DNA polymerase is convenient to use, can be directly used for multiple PCR amplification of target genes in samples with various sources and complex compositions after being matched with the PCR reaction buffer solution, and can normally amplify the target genes at least in a reaction system containing 50% of whole blood or 20% of milk.

(3) The qPCR reaction kit has the characteristics of strong specificity and high sensitivity, and can accurately detect the target gene with the concentration as low as 0.2 copies/mu L.

Drawings

FIG. 1 is a diagram showing the results of purification of Taq DNA polymerase; wherein, Lane 1 is (11.4-116.0kDa) wide range protein loading Marker, Lane 2 is supernatant obtained by centrifugation after cell disruption, Lane 3 is supernatant obtained by centrifugation after heating the supernatant of the disruption solution at 75 ℃ for 30min, Lane 4-9 are respectively Taq DNA polymerase in 10% -60% Buffer B linear elution peak during Ni-NTA affinity chromatography purification, and Lane 10 is Taq DNA polymerase obtained after anion exchange chromatography purification.

FIG. 2 is a graph showing the results of comparison of HS Sso-Taq of the present invention with a commercially available hot-start DNA polymerase TaqHS (Takara, R007Q) for direct amplification of whole blood; wherein A is an agarose gel electrophoresis image of PCR products obtained by amplifying human sterol 21-hydroxylase gene (CYP21) in whole blood at different concentrations by using two DNA polymerases; b is an agarose gel electrophoresis picture of a PCR product obtained by using the HS Sso-Taq for amplifying 3 DNA fragments with different lengths in 40% (v/v) whole blood.

FIG. 3 is a graph showing the results of agarose gel electrophoresis comparison of PCR products obtained when HS Sso-Taq of the present invention and a commercially available hot-start DNA polymerase TaqHS (Takara, R007Q) were used to directly amplify bovine mitochondrial pigment cell b (cob) genes in fresh milk at different concentrations.

FIG. 4 is a graph showing the comparison of the amplification sensitivity of a SYBR Green I dye-based fluorescent quantitative PCR reagent containing HS Sso-Taq with a commercially available reagent of the same type; wherein A is a result graph of a commercially available brand Promega (A600A), B is a result graph of a commercially available brand Takara (RR820), C is a result graph of HS Sso-Taq of the invention, and D is a result of amplification sensitivity and amplification efficiency of different reagents.

FIG. 5 is a graph showing the comparison of the performance of SYBR Green I fluorescent dye tolerance between HS Sso-Taq and hot-start wild-type Taq DNA polymerase (HS wt-Taq) according to the present invention; wherein A is an HS Sso-Taq amplification curve, B is an HS Sso-Taq melting curve, C is an HS wt-Taq amplification curve, and D is an HS wt-Taq melting curve.

FIG. 6 is a diagram showing the comparison result of humic acid tolerance of HS Sso-Taq and hot-start wild type Taq DNA polymerase (HS wt-Taq) in fluorescent quantitative PCR detection; wherein A is an HS Sso-Taq amplification curve, B is an HS Sso-Taq melting curve, C is an HS wt-Taq amplification curve, and D is an HS wt-Taq melting curve.

FIG. 7 is a graph showing the comparison of polysaccharide tolerance in fluorescent quantitative PCR detection between HS Sso-Taq and hot-start wild-type Taq DNA polymerase (HS wt-Taq) according to the present invention; wherein A is an HS Sso-Taq amplification curve, B is an HS Sso-Taq melting curve, C is an HS wt-Taq amplification curve, and D is an HS wt-Taq melting curve.

FIG. 8 is a graph showing the comparison of IgG tolerance performance of HS Sso-Taq and hot-start wild-type Taq DNA polymerase (HS wt-Taq) in fluorescent quantitative PCR detection. Wherein A is an HS Sso-Taq amplification curve, B is an HS Sso-Taq melting curve, C is an HS wt-Taq amplification curve, and D is an HS wt-Taq melting curve.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

EXAMPLE 1 construction of recombinant vector containing nucleotide sequence encoding Taq DNA polymerase

(1) According to the amino acid sequence (SEQ ID NO.1) of Taq DNA polymerase, after codon optimization of an escherichia coli expression system is carried out, a DNA molecule capable of being efficiently expressed in escherichia coli is obtained, and the DNA molecule for coding the Taq DNA polymerase is artificially synthesized by utilizing an overlap extension PCR method, wherein the DNA molecule is specifically shown as SEQ ID NO. 3.

(2) The DNA molecule encoding Taq DNA polymerase was subjected to homologous recombination with the expression vector pET-28 a. The Taq DNA polymerase amplification primer sequence is as follows:

Taq-FP：5’-GGCATATGGCGACCGTTAAGTTTAAGTAC-3’(SEQ ID NO.4)；

Taq-RP：5’-CCATGAATTCTTATTCCTTCGCAGAT-3’(SEQ ID NO.5)。

the pET-28a linearized primer sequence is as follows:

pET-28a-FP：5’-GGAATAAGAATTCATGGTTGCGGCCGCA-3’(SEQ ID NO.6)；

pET-28a-RP：5’-ACGGTCGCCATATGCCGCGCGGCACCA-3’(SEQ ID NO.7)。

PCR is used for amplifying Taq DNA polymerase DNA molecules and pET-28a vectors, the DNA molecules artificially synthesized and encoding the Taq DNA polymerase and pET-28a no-load are respectively used as templates, 25 mu L of 2 x Pfu Max HiFi PCR ProMix (Guangzhou Inzan Biotechnology Co., Ltd., product number P217A), 1 mu L (10 mu mol/L) of upstream and downstream primers and a proper amount of sterilized water are added for PCR amplification. The amplification conditions of the Taq DNA polymerase DNA molecule are as follows: 30s at 98 ℃; 30 cycles of 10s at 98 ℃, 30s at 55 ℃ and 1min at 68 ℃; 5min at 68 ℃. The plasmid linearization PCR amplification procedure was: 30s at 98 ℃; 30 cycles of 10s at 98 ℃, 30s at 55 ℃ and 2.5min at 68 ℃; 5min at 68 ℃. The Taq DNA polymerase DNA gene fragment of about 1.8kb in total length and the linearized pET-28a vector of about 5kb were recovered by agarose gel electrophoresis. The recovered products were subjected to homologous recombination in a system of 5. mu.L of 2 × Hipro DNA Assembly Cloning Mix (K001A Biotech, Guangzhou), 50ng each of the recovered products was supplemented with water to 10. mu.L, and incubated at 50 ℃ for 15 min. After incubation, the recombinant product was transformed into DH5a competent cells.

(3) Selecting a single colony for colony PCR identification, sending the positive single colony to a sequencing company for sequencing verification, culturing and verifying a correct competent cell, and extracting a plasmid, wherein the obtained plasmid is a recombinant vector containing a DNA molecule for encoding Taq DNA polymerase.

EXAMPLE 2 preparation of transformants expressing Taq DNA polymerase

The recombinant vector obtained in example 1 was transformed into a host cell E.coliBL21(DE3), and a single colony was picked up and inoculated into a liquid LB medium (containing 50. mu.g/mL kanamycin) to be cultured to OD ₆₀₀ And (3) adding IPTG (isopropyl thiogalactoside) to a final concentration of 0.1mmol/L, inducing at 37 ℃ for 3 hours, collecting thalli, carrying out ultrasonic disruption, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis to detect the expression of the target protein, and finding that the prepared transformant can efficiently express Taq DNA polymerase.

Example 3 expression of Taq DNA polymerase in recombinant E.coli

Inoculating the positive transformant strain capable of expressing Taq DNA polymerase obtained in example 2 into 60mL LB culture medium containing 50 ug/mL kanamycin, and shake-culturing overnight in a shaker at 37 ℃; taking out and culturing overnightThe seed solution of (1) was inoculated into 1L of LB medium containing 50. mu.g/mL kanamycin at a volume ratio of 1:100, and shake-cultured in a shaker at 37 ℃ to OD ₆₀₀ Is 0.7; adding IPTG into the shake flask until the final concentration is 0.1mmol/L, and continuing shaking and inducing for 3h at 37 ℃; after induction, the cells were collected by centrifugation and weighed, the wet weight of the cells was recorded and stored at-20 ℃.

Example 4 purification of Taq DNA polymerase

1. Ultrasonic crushing of recombinant thallus

Taking the induction expression thallus frozen at the temperature of 20 ℃ below zero, adding 5mL of lysis buffer (50mmol/L Tris-HCl, 100mmol/L NaCl, pH8.0) into each gram of thallus according to the wet weight of the thallus recorded in the example 3 for resuspension, and using an ultrasonic cell disruptor to lyse the thallus, wherein the ultrasonic conditions are as follows: the power is 250W, the ultrasound lasts for 5.5s, the stopping lasts for 5.5s, and the time lasts for 30 min. And putting the lysed thalli into a high-speed refrigerated centrifuge, centrifuging for 15min at the temperature of 4 ℃ at 20000r/min, and taking supernatant to a 250mL sterilized glass bottle. Incubating the supernatant in a constant temperature water bath at 75 deg.C for 30min, centrifuging at 4 deg.C for 15min at 20000r/min, filtering with 0.22 μm microporous membrane, and collecting supernatant in a 250mL sterilized glass bottle.

2. Nickel ion affinity chromatography purification

The chromatographic column is HisTrap ^TM HP 5mL (from GE Healthcare), binding buffer a: 50mmol/L Tris-HCl, 50mmol/L NaCl, pH 8.0; the elution buffer was buffer B: 500mmol/L imidazole, 50mmol/L Tris-HCl, 50mmol/L NaCl, pH8, 0.22 μm filter membrane filtration for use.

HisTrap ^TM And (2) putting HP 5mL into a column position valve of a rapid protein purification instrument, then washing a system and a column with ultrapure water, balancing the column with buffer A, then loading the supernatant obtained in the step (1) with a sample pump, washing the column with buffer A after loading is finished, then performing linear elution (10% -60%) with buffer B, and collecting a linear elution peak sample. Samples of 1-6 tubes (3 mL/tube) collected from the buffer B elution peak were sampled and detected by SDS-PAGE protein electrophoresis, and the results are shown in FIG. 1 (lanes 4-9), and samples of 5 and 6 tubes with higher purity were combined to be used as the strong anion exchange column chromatography samples.

3. Strong anion exchange column chromatography

The chromatographic column is HisTrap ^TM Capto ^TM Q5 mL (purchased from GE Healthcare), the binding Buffer used was Buffer C: 50mmol/L Tris-HCl, 100mmol/L NaCl, pH 8.0; the elution buffer was BufferD: 50mmol/L Tris-HCl, 1mol/L NaCl, pH8.0, 0.22 μm filter membrane for use.

HisTrap ^TM Capto ^TM And (3) inserting the Q5 mL into a column position valve of the AKTA purifier, then cleaning a system and a column by using ultrapure water, balancing the column by using buffer C, then loading the affinity chromatography sample containing the target protein by using a sample pump, after the loading is finished, firstly cleaning the column by using the buffer C, then performing gradient elution by using an elution buffer solution buffer D, and collecting an elution peak. The eluted peak was sampled at 20. mu.L and the results of SDS-PAGE protein electrophoresis were shown in FIG. 1.

Example 5 Taq DNA polymerase Activity assay

The enzyme activity of Taq DNA polymerase prepared in example 4 was measured in the following manner. At 74 ℃, using the activated salmon sperm DNA as a template/primer, and adding 200 mu mol/L dNTPs, 50mmol/L Tris-HCl and 2mmol/L MgCl ₂ 、5mmol/L(NH ₄ ) ₂ SO ₄ 50mmol/L KCl, 0.1-0.4 mg/L BSA, 0.1% Tween-20 and 4% DMSO, and the enzyme amount for catalyzing 10nmoldNTPs to be doped into DNA within 30min is defined as 1U. The result shows that the enzyme activity concentration of the TaqDNA polymerase is 62U/. mu.L.

Example 6 preparation and Activity measurement of HS Sso-Taq

Dialyzing the high-purity Taq DNA polymerase into 10-30 mmol/L Tris-HCl (pH9.0-10.0) buffer solution overnight. And measuring the protein concentration by using a BCA method, uniformly mixing the protein concentration and the citric acid chaff with anhydride according to the molar ratio of 1:100, and incubating for 4 hours at 42 ℃ to obtain the hot-start DNA polymerase with reversible blocking enzyme activity modified by the citric acid chaff, which is named as HS Sso-Taq.

The resulting hot start DNA polymerase was dialyzed against storage buffer (20mmol/L Tris-HCl, 100mmol/L KCl, 0.1mmol/L EDTA, 50% Glycerol, 1mmol/L DTT, 0.5% Tween-20, 0.5%

CA-630, pH8.0), 95 ℃ and 10min after heat shock, the activity of the hot-start DNA polymerase was determined as described for the activity assay in example 5. The result shows that the enzyme activity concentration of HS Sso-Taq is 5U/. mu.L.

Example 7 Whole blood direct expansion Capacity study of HS Sso-Taq

(1) Direct amplification of whole blood at various concentrations

The results of amplifying the human sterol 21-hydroxylase gene (CYP21) gene simultaneously with the direct amplification type hot-start DNA polymerase of the present invention and the commercially available hot-start DNA polymerase TaqHS (Takara, R007Q) of Takara, which is a brand name, were shown in FIG. 2, using 100ng of human genomic DNA purified from EDTA-2Na anticoagulated blood and 10%, 20%, 40%, 50% (v/v) EDTA-2Na anticoagulated blood (whole blood was provided by healthy persons, sampled in Guangdong province), respectively, as templates. The amplification primers and the specific operation are as follows:

CYP21-FP：5’-GCTCAGCATGGTGGTGGCATAA-3’(SEQ ID NO.8)；

CYP21-RP：5’-CCTCATACCTTCCCCCCCATTT-3’(SEQ ID NO.9)。

the PCR reaction buffer according to the present invention comprises 50mmol/L Tris-HCl (pH8.0), 3mmol/L MgCl ₂ 、5mmol/L(NH ₄ ) ₂ SO ₄ 50mmol/L KCl, 0.1mg/L BSA, 0.05% Tween-20, 1% DMSO, 250nmol/L CYP21-FP, 250nmol/L CYP21-RP, 200 μmol/L dNTPs, 0.05U/μ L HS Sso-Taq, and a proper amount of sterilized ddH ₂ And O. The reaction program is 95 ℃ for 10 min; 35 cycles of 95 ℃ for 15s, 60 ℃ for 30s and 72 ℃ for 30 s.

A commercially available reaction system of Takara Hot Start DNA polymerase Taq HS brand comprises 2.5. mu.L of 10 XPCR Buffer, 200nmol/L CYP21-FP, 200nmol/L CYP21-RP, 200. mu. mol/L dNTPs, 0.05U/. mu.L Taq HS, and a proper amount of sterilized ddH ₂ And O. The reaction sequence was 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 30s for 35 cycles.

The results are shown in FIG. 2A. As a result of the analysis, the direct amplification type hot-start DNA polymerase of the present invention can tolerate at least 50% of whole blood and amplify a gene of interest therefrom. Whereas the commercial hot start DNA polymerase TaqHS is not tolerant to inhibition by whole blood.

(2) Amplification of 3 gene fragments of different lengths in 40% whole blood

The direct amplification type hot start DNA polymerase is used for simultaneously amplifying gene segments with different lengths by taking 40% EDTA-2Na anticoagulation as a template, and the amplification genes, primers and specific operations are as follows:

human sterol 21-hydroxylase gene (CYP21) with gene length of 320bp, and primers shown in SEQ ID NO.8 and SEQ ID NO. 9.

Diacylglycerol kinase (DGK) gene, the length of which is 243bp,

DGK-FP：5’-GGAACAAGACACGGCTGGGTT-3’(SEQ ID NO.10)；

DGK-RP：5’-AGCAAGGCAGGGCAGGCAAGT-3’(SEQ ID NO.11)。

bHLH transcription factor gene (bHLH transcription factor, bHLH TF), the gene length is 100bp,

bHLH TF-FP：5’-GTCCTTCCCCCGCTGGAAAC-3’(SEQ ID NO.12)；

bHLH TF-RP：5’-GCAGCAGAGATCATCGCGCC-3’(SEQ ID NO.13)。

the PCR reaction buffer according to the present invention comprises 50mmol/L Tris-HCl (pH8.0), 3mmol/L MgCl ₂ 、5mmol/L(NH ₄ ) ₂ SO ₄ 50mmol/L KCl, 0.1mg/L BSA, 0.05% Tween-20, 1% DMSO, 200nmol/L CYP21-FP/CYP21-RP, DGK-FP/DGK-RP, bHLH TF-FP/bHLH TF-RP, 200 μmol/L dNTPs, 0.05U/μ L HS Sso-Taq, a proper amount of template, and a proper amount of sterilized ddH ₂ And O. The reaction program is 95 ℃ for 10 min; 35 cycles of 95 ℃ for 15s, 60 ℃ for 30s and 72 ℃ for 30 s.

The results are shown in FIG. 2B. According to the analysis result, the direct amplification type hot start DNA polymerase can simultaneously amplify three gene fragments (320bp, 243bp and 100bp) with different lengths in a PCR system with the whole blood content of 40% (v/v). This is the first time that Taq DNA polymerase completes amplification of three genes simultaneously in 40% (v/v) whole blood.

Example 8 milk straight-extending ability test of HS Sso-Taq

Using 2ng of genomic DNA purified from fresh milk and 5%, 10%, 15%, 20% (v/v) of fresh milk (Guangming-excellent times) as templates, the bovine mitochondrial pigment cell b (cob) gene was amplified simultaneously with the direct amplification type hot-start DNA polymerase of the present invention and the hot-start DNA polymerase HS-Taq purchased from Takara, a amplification primer having an amplification length of 424bp and the following operations:

cob-FP：5’-AAGACGAGAAGACCCTATGGAGCTTTA-3’(SEQ ID NO.14)；

cob-RP：5’-GATTGCGCTGTTATCCCTAGGGTA-3’(SEQ ID NO.15)。

the PCR reaction buffer according to the present invention comprises 50mmol/L Tris-HCl (pH8.0), 3mmol/L MgCl ₂ 、5mmol/L(NH ₄ ) ₂ SO ₄ 50mmol/L KCl, 0.1mg/L BSA, 0.05% Tween-20, 1% DMSO, 200 nmol/Lcobs-FP, 200 nmol/Lcobs-RP, 200 μmol/L dNTPs, 0.05U/μ L HS Sso-Taq, and a proper amount of sterilized ddH ₂ And O. The reaction program is 95 ℃ for 10 min; 35 cycles of 95 ℃ for 15s, 60 ℃ for 30s and 72 ℃ for 30 s.

A commercially available brand Takara Hot Start DNA polymerase TaqHS reaction system comprises 2.5. mu.L 10 XPCR Buffer, 200nmol/Lcob-FP, 200nmol/Lcob-RP, 200. mu. mol/L dNTPs, 0.05U/. mu.L TaqHS, and an appropriate amount of sterile ddH ₂ And O. The reaction sequence was 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 30s for 35 cycles.

The results are shown in FIG. 3. As a result of analysis, the direct-amplification-type hot-start DNA polymerase of the present invention can tolerate at least 20% of fresh milk, and the target gene is amplified therefrom. While the commercial hot start DNA polymerase TaqHS can only tolerate 10% of fresh milk. Compared with the commercial hot start DNA polymerase, the hot start DNA polymerase has stronger direct amplification capability.

Example 9 HS Sso-Taq based sensitivity test of qPCR reaction reagents of the present invention

The recombinant internal reference gene U6 plasmid (U6-pMD 18-T, the inserted position of B2M is restriction enzyme site Ecor V) diluted by gradient is taken as a template, the copy number concentration of the recombinant plasmid in the last gradient is about 0.2 copies/mu L, the U6 gene is amplified simultaneously by qPCR reaction liquid and purchased products of the same type of commercial brands Takara and Promega, and the amplification primers and the specific operation are as follows:

U6-FP：5’-GCTCGCTTCGGCAGCACATAT-3’(SEQ ID NO.16)；

U6-RP：5’-CGCTTCACGAATTTGCGTGTC-3’(SEQ ID NO.17)。

the reaction system based on the qPCR reaction solution comprises 0.05U/. mu.L HS Sso-Taq, 200. mu. mol/L dNTPs, 50mmol/L Tris-HCl (pH8.0), and 3mmol/L MgCl ₂ 、5mmol/L(NH ₄ ) ₂ SO ₄ 50mmol/L KCl, 0.1mg/L BSA, 0.05% Tween-20, 4% DMSO, 0.4 XSSYBR Green I, 250nmol/L U6-FP, 250nmol/L U6-RP, 1 μ L template, appropriate amount of sterilized ddH ₂ O, total volume of reaction was 20. mu.L. The reaction program is 95 ℃ and 10 min; 5s at 95 ℃, 20s at 60 ℃ and read plate for 40 cycles; the melting curve is read.

Commercially available Takara congener reagent TB

Premix Ex Taq ^TM II (TliRNaseH plus) reaction system containing 10. mu.L of 2

Premix Ex Taq ^TM II, 400nmol/L U6-FP, 400nmol/L U6-RP, 1 μ L template, appropriate amount of sterilized ddH ₂ O, 20 μ L in total. The reaction program is 95 ℃ for 30 s; 5s at 95 ℃, 20s at 60 ℃ and read plate for 40 cycles; the melting curve is read.

Commercially available Promega congener reagents

The reaction system of qPCR Master Mix contains 10. mu.L 2

qPCR Master Mix, 400nmol/L U6-FP, 400nmol/L U6-RP, 1 μ L template, sterilized ddH in appropriate amounts ₂ O, total reaction volume 20. mu.L. The reaction program is 95 ℃ for 2 min; the melting curve was read at 95 ℃ for 3s, 60 ℃ for 30s, and read plate for 40 cycles.

The results are shown in FIG. 4. The result of the analysis can be obtained, the qPCR reaction reagent can accurately detect the target gene with the concentration of 0.2 copies/mu L, has excellent sensitivity, the amplification efficiency is close to 100 percent, and the linear relation between gradients is 0.999, so that accurate quantification and trace nucleic acid detection can be carried out. Compared with the commercially available Promega (A600A) and Takara (RR820), the method has higher sensitivity and amplification efficiency.

Example 10 HS Sso-Taq tolerance inhibitor Performance Studies in qPCR assays

1ng of recombinant internal reference gene B2M plasmid (purchased, B2M-pMD 18-T, B2M inserted position is restriction enzyme cleavage site Ecor V) is used as a template, and the B2M gene is amplified by using equal amounts of HS wt-Taq (hot start wild type Taq DNA polymerase) and HS Sso-Taq in the presence of different inhibitors, wherein the amplification primers are as follows:

B2M-FP：5’-CTATCCAGCGTACTCCAAAG-3’(SEQ ID NO.18)，

B2M-RP：5’-GAAAGACCAGTCCTTGCTGA-3’(SEQ ID NO.19)。

each inhibitor test qPCR reaction system contained the following general components: 0.05U/. mu.L HS wt-Taq/HS Sso-Taq, 200nmol/L B2M-FP, 200nmol/L B2M-RP, 200. mu. mol/L dNTPs, 50mmol/L Tris-HCl, 2mmol/L MgCl ₂ 、5mmol/L(NH ₄ ) ₂ SO ₄ 30-80 mmol/L KCl, 0.1mg/L BSA, 0.05% Tween-20, 3% DMSO, 0.3mol/L trehalose, 0.2mol/L L-carnitine and 0.4% NP40, wherein the pH of the reaction system is 8.0.

(1) SYBR Green I tolerance performance test

Respectively adding SYBR Green I with final reaction concentrations of 0 ng/mu L, 0.5 ng/mu L, 1.0 ng/mu L, 2.0 ng/mu L, 4.0 ng/mu L, 8.0 ng/mu L and 10.0 ng/mu L into the general components, wherein the reaction procedures are all 10min at 95 ℃; 5s at 95 ℃, 20s at 60 ℃ and read plate for 40 cycles; the melting curve is read. The result is shown in fig. 5, and it can be seen from the result that HS wt-Taq can tolerate a high concentration dye with a maximum SYBR Green la concentration of 1.0ng/μ L, and HS Sso-Taq can tolerate a high concentration dye with a reaction concentration of at least 10.0ng/μ L, and HS Sso-Taq has a stronger SYBR Green la tolerance ability than HS wt-Taq.

(2) Humic acid resistance test

Respectively adding 0.4 XSSYBR Green I (HS wt-Taq) and 0.8 XSSYBR Green I (HS Sso-Taq) into the general components, and respectively adding humic acid with final concentrations of 0 mug/mL, 0.1 mug/mL, 0.2 mug/mL, 0.4 mug/mL, 0.8 mug/mL and 1.6 mug/mL, wherein the reaction process is 95 ℃ for 10 min; 5s at 95 ℃, 20s at 60 ℃ and read plate for 45 cycles; the melting curve is read. The results are shown in FIG. 6, and it can be seen from the results that HS Sso-Taq can tolerate humic acid with the final reaction concentration of 1.6. mu.g/mL, and the humic acid inhibition resistance of wild type Taq DNA polymerase is greatly improved.

(3) Tolerance polysaccharide Performance test

Respectively adding astragalus polysaccharide with final reaction concentrations of 0.4 xSYBR Green I (HS wt-Taq) and 0.8 xSYBR Green I (HS Sso-Taq) into the general components, and respectively adding astragalus polysaccharide with final reaction concentrations of 0mg/mL, 0.02mg/mL, 0.04mg/mL, 0.08mg/mL, 0.16mg/mL and 0.32mg/mL, wherein the reaction procedures are all 10min at 95 ℃; 5s at 95 ℃, 20s at 60 ℃ and read plate for 45 cycles; the melting curve is read. As shown in FIG. 7, it can be seen that HS Sso-Taq can tolerate astragalus polysaccharides up to a reaction concentration of 0.32mg/mL, while wild-type Taq DNA polymerase can tolerate astragalus polysaccharides up to a reaction concentration of 0.08 mg/mL.

(4) Immunoglobulin IgG resistance assay

Respectively adding 0.4 XSSYBR Green I (HS wt-Taq) and 0.8 XSSYBR Green I (HS Sso-Taq) into the general components, and respectively adding immunoglobulin IgG with final concentrations of 0 mug/mL, 75 mug/mL, 150 mug/mL, 300 mug/mL and 600 mug/mL, wherein the reaction procedures are 95 ℃ and 10 min; 95 ℃ for 5s, 60 ℃ for 20s, read plate; a total of 45 cycles; the melting curve is read. As a result, as shown in FIG. 8, it was found that HS Sso-Taq can tolerate immunoglobulin IgG up to a reaction concentration of 600. mu.g/mL, and wild-type Taq DNA polymerase hardly had the anti-IgG inhibitory activity.

In conclusion, the hot-start DNA polymerase has excellent direct amplification capability and stronger qPCR inhibitor resistance capability, and is suitable for crude direct fluorescence quantitative PCR.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> south China university of science and technology

<120> direct amplification type hot start DNA polymerase, preparation method and application thereof

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 631

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> direct amplification type hot start DNA polymerase amino acid sequence

<400> 1

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu

20 25 30

Lys Glu Val Asp Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys

35 40 45

Met Ile Ser Phe Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly

50 55 60

Ala Val Ser Glu Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu

65 70 75 80

Lys Gln Lys Lys Gly Gly Val Thr Ser Pro Lys Ala Leu Glu Glu Ala

85 90 95

Pro Trp Pro Pro Pro Glu Gly Ala Phe Val Gly Phe Val Leu Ser Arg

100 105 110

Lys Glu Pro Met Trp Ala Asp Leu Leu Ala Leu Ala Ala Ala Arg Gly

115 120 125

Gly Arg Val His Arg Ala Pro Glu Pro Tyr Lys Ala Leu Arg Asp Leu

130 135 140

Lys Glu Ala Arg Gly Leu Leu Ala Lys Asp Leu Ser Val Leu Ala Leu

145 150 155 160

Arg Glu Gly Leu Gly Leu Pro Pro Gly Asp Asp Pro Met Leu Leu Ala

165 170 175

Tyr Leu Leu Asp Pro Ser Asn Thr Thr Pro Glu Gly Val Ala Arg Arg

180 185 190

Tyr Gly Gly Glu Trp Thr Glu Glu Ala Gly Glu Arg Ala Ala Leu Ser

195 200 205

Glu Arg Leu Phe Ala Asn Leu Trp Gly Arg Leu Glu Gly Glu Glu Arg

210 215 220

Leu Leu Trp Leu Tyr Arg Glu Val Glu Arg Pro Leu Ser Ala Val Leu

225 230 235 240

Ala His Met Glu Ala Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Arg

245 250 255

Ala Leu Ser Leu Glu Val Ala Glu Glu Ile Ala Arg Leu Glu Ala Glu

260 265 270

Val Phe Arg Leu Ala Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln

275 280 285

Leu Glu Arg Val Leu Phe Asp Glu Leu Gly Leu Pro Ala Ile Gly Lys

290 295 300

Thr Glu Lys Thr Gly Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala

305 310 315 320

Leu Arg Glu Ala His Pro Ile Val Glu Lys Ile Leu Gln Tyr Arg Glu

325 330 335

Leu Thr Lys Leu Lys Ser Thr Tyr Ile Asp Pro Leu Pro Asp Leu Ile

340 345 350

His Pro Arg Thr Gly Arg Leu His Thr Arg Phe Asn Gln Thr Ala Thr

355 360 365

Ala Thr Gly Arg Leu Ser Ser Ser Asp Pro Asn Leu Gln Asn Ile Pro

370 375 380

Val Arg Thr Pro Leu Gly Gln Arg Ile Arg Arg Ala Phe Ile Ala Glu

385 390 395 400

Glu Gly Trp Leu Leu Val Ala Leu Asp Tyr Ser Gln Ile Glu Leu Arg

405 410 415

Val Leu Ala His Leu Ser Gly Asp Arg Asn Leu Ile Arg Val Phe Gln

420 425 430

Glu Gly Arg Asp Ile His Thr Glu Thr Ala Ser Trp Met Phe Gly Val

435 440 445

Pro Arg Glu Ala Val Asp Pro Leu Met Arg Arg Ala Ala Lys Thr Ile

450 455 460

Asn Phe Gly Val Leu Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu

465 470 475 480

Leu Ala Ile Pro Tyr Glu Glu Ala Gln Ala Phe Ile Glu Arg Tyr Phe

485 490 495

Gln Ser Phe Pro Lys Val Arg Ala Trp Leu Arg Lys Thr Leu Glu Glu

500 505 510

Gly Arg Arg Arg Gly Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr

515 520 525

Val Pro Asp Leu Glu Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu

530 535 540

Arg Met Ala Phe Asn Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met

545 550 555 560

Lys Leu Ala Met Val Lys Leu Phe Pro Arg Leu Glu Glu Met Gly Ala

565 570 575

Arg Met Leu Leu Gln Val His Asp Glu Leu Val Leu Glu Ala Pro Lys

580 585 590

Glu Arg Ala Glu Ala Val Ala Arg Leu Ala Lys Glu Val Met Glu Gly

595 600 605

Val Tyr Pro Leu Ala Val Pro Leu Glu Val Glu Val Gly Ile Gly Glu

610 615 620

Asp Trp Leu Ser Ala Lys Glu

625 630

<210> 2

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Sso7d amino acid sequence

<400> 2

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His

20

<210> 3

<211> 1893

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> direct amplification type hot start DNA polymerase nucleotide sequence

<400> 3

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggcgacgg ttaaattcaa gtataagggc gaggagaaag aagtagatat tagcaagatt 120

aagaaagtgt ggcgcgtagg caaaatgatt tctttcacct atgatgaagg aggtggcaaa 180

accggtcgtg gggcggtgtc agaaaaggat gcgccgaagg aacttcttca aatgctggag 240

aaacagaaga agggcggagt gaccagcccg aaagcccttg aggaggcacc atggccaccg 300

cctgaaggtg cgtttgtggg gtttgtgctg agccgtaagg aaccgatgtg ggccgatctg 360

ttggcattgg cagccgcacg tggtggccgc gtgcatcgcg ctccagaacc gtacaaagcg 420

ctgcgggatc tgaaagaggc tcgtggatta ctggcgaagg atcttagcgt gcttgctctt 480

cgcgagggcc tgggtcttcc acctggtgac gatccgatgc ttcttgcata tctgctggat 540

ccgagcaaca ccaccccgga gggcgtggct cgtcggtatg gaggtgaatg gaccgaagaa 600

gccggagaac gtgccgcgtt aagcgagcgg ctgtttgcga atctttgggg tcgtcttgag 660

ggggaagagc gtctgctgtg gctttatcgt gaagtcgagc gtccgcttag tgcagtgtta 720

gcgcacatgg aggccacggg tgttcgttta gacgtggcgt acctgcgtgc gttgagcctg 780

gaagtggcgg aggaaatagc gcggttggag gcggaagttt ttcgcttggc gggtcaccct 840

tttaatctga atagccgtga tcagctggaa cgtgtcctgt tcgatgaact gggcctgccg 900

gccattggga agacagagaa aacaggcaaa cgttcaacca gcgcggcagt gcttgaagct 960

ttacgtgagg cccacccaat tgtggaaaaa atcctgcagt atcgcgaact gaccaaactg 1020

aaaagcacct acattgatcc gctgccggac ctgattcacc cgcgtaccgg acgcctgcat 1080

acccgtttca atcagaccgc gaccgccaca ggccggttga gtagcagcga tcccaactta 1140

cagaacattc cggtgcgtac cccgttaggc cagcgcattc gccgtgcctt tattgcggaa 1200

gagggctggc tgcttgtggc acttgattat agccaaattg aactgcgtgt gttggctcac 1260

ctgtctggcg atcggaacct tatccgggta tttcaggagg gacgtgatat tcatacagaa 1320

accgcgagct ggatgttcgg tgtaccgcgc gaagcagtag atcctctgat gcggcgtgct 1380

gctaagacaa tcaattttgg cgtgttatat ggcatgtccg cccatcgtct gagtcaagaa 1440

ttggcaatac cgtacgaaga ggcgcaggca tttatcgagc ggtattttca gagttttccg 1500

aaagttcggg catggctgcg taaaaccctg gaggaaggtc gccgtcgggg ctacgttgag 1560

actctgtttg ggcgtcggcg ttatgttccg gatctggaag cgcgtgtgaa atccgtgcgg 1620

gaagcagctg aacgcatggc ctttaacatg cctgtgcagg ggaccgccgc tgatctgatg 1680

aaactggcta tggtgaaact tttcccacgc ttggaggaga tgggggctcg tatgttgctg 1740

caggtccatg acgaactggt tttagaagct ccaaaggaac gcgcagaggc agttgcgcgc 1800

ctggctaagg aagttatgga aggtgtgtat ccgttagcgg tgccgctgga ggttgaagtg 1860

ggcataggag aggattggct gagtgcaaaa gaa 1893

<210> 4

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Taq -FP

<400> 4

ggcatatggc gaccgttaag tttaagtac 29

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Taq -RP

<400> 5

ccatgaattc ttattccttc gcagat 26

<210> 6

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pET-28a-FP

<400> 6

ggaataagaa ttcatggttg cggccgca 28

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pET-28a-RP

<400> 7

acggtcgcca tatgccgcgc ggcacca 27

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CYP21-FP

<400> 8

gctcagcatg gtggtggcat aa 22

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CYP21-RP

<400> 9

cctcatacct tcccccccat tt 22

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> DGK -FP

<400> 10

ggaacaagac acggctgggt t 21

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> DGK -RP

<400> 11

agcaaggcag ggcaggcaag t 21

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> bHLH TF -FP

<400> 12

gtccttcccc cgctggaaac 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> bHLH TF -RP

<400> 13

gcagcagaga tcatcgcgcc 20

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> cob-FP

<400> 14

aagacgagaa gaccctatgg agcttta 27

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> cob-RP

<400> 15

gattgcgctg ttatccctag ggta 24

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> U6-FP

<400> 16

gctcgcttcg gcagcacata t 21

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> U6-RP

<400> 17

cgcttcacga atttgcgtgt c 21

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M-FP

<400> 18

ctatccagcg tactccaaag 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B2M-RP

<400> 19

gaaagaccag tccttgctga 20

Claims

1. A direct-amplification hot-start DNA polymerase, comprising: the amino acid sequence is shown in SEQ ID NO. 1.

2. A DNA molecule encoding the direct-amplification hot-start DNA polymerase of claim 1, wherein: the nucleotide sequence is shown in SEQ ID NO. 3.

3. A recombinant expression vector characterized by: is obtained by cloning a DNA molecule encoding the direct-amplification hot-start DNA polymerase of claim 1 into an expression vector.

4. A recombinantly engineered cell strain characterized by: is obtained by transforming the recombinant expression vector of claim 3 into an engineered cell.

5. The direct-amplification hot-start DNA polymerase according to claim 1, wherein:

the polymerase active site lysine side chain amino of the direct amplification type hot start DNA polymerase is specifically combined with an anhydride compound; the anhydride compound is further citric acid anhydride.

6. The method for preparing a direct-amplification hot-start DNA polymerase according to claim 1 or 5, wherein: the method comprises the following steps:

inoculating the recombinant engineered cell strain of claim 4 into LB culture medium containing antibiotics, and culturing at 35-38 deg.C to OD ₆₀₀ Reaching 0.7-0.9, inducing cells to express proteins by IPTG, and separating and purifying to obtain the direct-amplification hot start DNA polymerase;

when the acid anhydride compound is specifically combined on the lysine side chain amino group of the polymerase active site of the direct amplification type hot start DNA polymerase, the preparation method further comprises the following steps:

(2) adding an anhydride compound, uniformly mixing, and incubating;

(3) dialyzing the incubated enzyme solution into a storage buffer solution to obtain the stable direct-amplification hot-start DNA polymerase.

7. The method for preparing a direct-amplification-type hot-start DNA polymerase according to claim 6, wherein:

the dosage of the IPTG is calculated according to the final concentration of 0.1-0.5 mmol/L;

the induction conditions are that the temperature is 35-38 ℃ and the time is 2-4 h;

the separation and purification method comprises the steps of performing nickel ion affinity chromatography and then performing anion exchange column chromatography;

the Tris-HCl buffer solution is a Tris-HCl buffer solution with the concentration of 10-30 mmol/L, pH = 9-10;

the proportion of the direct amplification type hot start DNA polymerase to the anhydride compound is 1: 50-1: 100 in molar ratio;

the incubation condition is that the temperature is 40-50 ℃ and the time is 2-5 h;

the storage buffer solution comprises the following formula: 20mmol/L Tris-HCl, 100mmol/L KCl, 0.1mmol/L EDTA, 50% glycerol, 1mmol/L DTT, 0.5% Tween-20, 0.5% IGEPAL CA-630 and pH8.0.

8. A qPCR reaction kit, characterized in that: comprising at least one of PCR reaction buffer, SYBR Green I, water for PCR and dNTPs, and the direct-amplification-type hot-start DNA polymerase described in claim 1 or 5;

the concentration of the SYBR Green I in the system is 0.2-10 ng/mu L;

the concentration of the direct amplification type hot start DNA polymerase in the system is 0.02-0.05U/mu L;

the concentration of the dNTPs in the system is 100-300 mu mol/L.

9. The qPCR reaction kit according to claim 8, characterized in that:

the PCR reaction buffer comprises the following components: 20 to 100mmol/L Tris-HCl, 1 to 3mmol/L MgCl ₂ 、5～20 mmol/L (NH ₄ ) ₂ SO ₄ 20-80 mmol/L KCl, 0.1-0.4 mg/L BSA, 0.05-0.1% Tween-20, 1-6% DMSO, and the pH value is 8-10;

the PCR reaction buffer solution also comprises at least one of trehalose, ethyl phenyl polyethylene glycol and L-carnitine;

the concentration of the trehalose in the system is 0.1-0.4 mol/L;

the concentration of the ethyl phenyl polyethylene glycol in the system is 0-0.4%;

the concentration of the L-carnitine in the system is 0.1-0.3 mol/L.

10. Use of a qPCR reaction kit according to claim 8 or 9 in the amplification of a DNA sample.