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Preparation method and application of DNA polymerase accelerant

A polymerase and accelerator technology, which is applied in the fields of protein engineering and nucleic acid amplification, can solve the problems of lack of calibration activity of A-type DNA polymerase and low fidelity of amplified DNA, and achieves improved fidelity, strong versatility, and elimination of The effect of base mutations

Inactive Publication Date: 2017-05-31
SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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AI Technical Summary

Problems solved by technology

Type A DNA polymerase has no proofreading activity, and the fidelity of amplified DNA is much lower than that of type B DNA polymerase

Method used

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Examples

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Embodiment

[0014] Utilizing UDG and AlkA DNA glycosidase and endonuclease IV of Pyrococcus to prepare DNA polymerase accelerator and its application in PCR catalyzed by Pfu DNA polymerase.

[0015] The first step is the preparation of Pyrococcus UDG. The Pyrococcus udg gene was cloned into the prokaryotic expression vector pET28a, the cloning sites were NdeI and BamHI, and the recombinant expression vector pET28a-udg of Pyrococcus UDG was constructed. Then the expression vector pET28a-udg was transformed into E. coli expression strain BL21 (DE3). When E. coli grew to OD600=0.5, IPTG with a final concentration of 0.5mM was added to induce the expression of Pyrococcus UDG protein. Finally, the IPTG-induced E. coli was sonicated, and the expressed Pyrococcus UDG was purified by nickel ion affinity chromatography.

[0016] The second step is the preparation of Pyrococcus AlkA. The preparation method of Pyrococcus thermostable DNA glycosidase AlkA is similar to that of thermostable UDG, exc...

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Abstract

The invention discloses a preparation method and application of a DNA polymerase accelerant. The DNA polymerase accelerant is composed of three kinds of heat-stability protein which are uracil DNA glycosidase UDG, hypoxanthine DNA glycosidase AlkA and endonuclease IV respectively. UDG is in charge of cutting off dU basic groups generated in a PCR, AlkA is in charge of cutting off generated dI basic groups, and endonuclease IV is in charge of cutting off basic group removal loci. The joint action of the three kinds of protein can achieve the purposes that the inhibition effect of the dU / dI basic groups on the activity of B type DNA polymerase is released; the dU / dI basic groups are repaired and corrected, and the PCR faithfulness of the A type and B type DNA polymerase is improved. In addition, the activity of 3'-excision enzymes of endonuclease IV can provide deleted correction activity of the A type DNA polymerase. The DNA polymerase accelerant can effectively increase the PCR yield of the B type DNA polymerase by 1-3 times.

Description

technical field [0001] The invention belongs to the technical fields of protein engineering and nucleic acid amplification, and in particular relates to a preparation method and application of a DNA polymerase accelerator. Background technique [0002] Polymerase chain reaction (PCR) is a molecular biology technique widely used in life science research to amplify and detect various nucleic acid fragments. Various high-temperature resistant DNA polymerases are widely used in PCR, mainly divided into A-type and B-type DNA polymerases. Type A DNA polymerase has no proofreading activity, and the fidelity of amplified DNA is much lower than that of type B DNA polymerase. B-type DNA polymerase has proofreading activity and amplifies DNA with high fidelity. PCR is carried out at high temperature, which leads to the deamination of dC and dA bases in DNA to generate dU and dI bases. B-type DNA polymerase has a high binding force to dU and dI bases in DNA, causing the DNA synthesis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/22C12N9/24
CPCC12N9/1252C12N9/22C12N9/2497C12Y207/07007C12Y302/02002C12Y302/02003
Inventor 刘喜朋
Owner SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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