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Pfu DNA polymerase

A polymerase and gene technology, applied in the field of bioengineering, can solve the problems of slow extension speed and weak stability of PfuDNA polymerase, and achieve the effects of unchanged amplification speed and fidelity, improved stability, and increased recovery rate.

Active Publication Date: 2022-02-08
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the above technical problems, the present invention provides a Pfu DNA polymerase, which solves the problems of slow elongation and weak stability of the existing Pfu DNA polymerase. In addition, the new Pfu DNA enzyme of the present invention adds 8 His label, which greatly facilitates subsequent purification steps

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Embodiment Construction

[0032] The present invention will be further described below in conjunction with specific examples, so that those skilled in the art can better understand the present invention and implement it, but the given examples are not intended to limit the present invention.

[0033] What the present invention adopts are conventional test methods, and materials and reagents are all commercially available products.

[0034] The pET-30(a)+ plasmid was digested with EcoRI and NotI to obtain a large plasmid fragment. After 3-4 hours in a metal bath at 37°C, it was detected by 1% agarose gel electrophoresis, and finally an agarose gel recovery kit was used. Recovering to obtain carrier fragments;

[0035] Ligate the gene fragment encoding Pfu DNA polymerase and the vector fragment, overnight at 16°C, transfer the constructed recombinant plasmid into Ecoli BL21(DE3), and obtain the expression strain;

[0036] Fill 200 mL of LB medium in a 1 L liquid shake flask, add 100 μg / mL of kanamycin, ...

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Abstract

The invention relates to the technical field of bioengineering, in particular to Pfu DNA polymerase, wherein the amino acid sequence of the Pfu DNA polymerase is shown as Seq ID No.1, the concentration of dialyzed protein is 1.2 mg / mL, the nucleotide sequence for coding the gene of the Pfu DNA polymerase is shown as Seq ID No.2, an expression vector carrying the gene is transferred into host bacteria to construct an expression strain, and the Pfu DNA polymerase is obtained through induced expression. According to the invention, the Pfu DNA polymerase can amplify DNA fragments within 6000bp without mismatch, and has high fidelity, high extension speed of 42bp / s and higher denaturation temperature of 100 DEG C; a relatively complete protein structure and an excellent amplification effect can still be kept after the enzyme is stored at 37 DEG C for one week, and the stability is remarkably improved compared with that of a wild type unmodified enzyme; and the Pfu DNA polymerase disclosed by the invention can save more time in the PCR reaction, is higher in stability, and increases the recovery efficiency after purification.

Description

technical field [0001] The invention relates to a Pfu DNA polymerase, belonging to the technical field of bioengineering. Background technique [0002] PCR manipulation is the most basic and common technique in molecular biology. The PCR reaction is catalyzed by a DNA polymerase, and selecting a suitable polymerase according to the purpose of the experiment is a problem that needs to be considered before performing a PCR experiment. The main difference between many DNA polymerases lies in several indicators such as specificity, fidelity, amplification rate and amplified fragment length. [0003] Currently the most commonly used DNA polymerase is Taq enzyme, which has high amplification efficiency but no correction function. Pfu enzyme has excellent thermostability, has 5'-3' DNA polymerase activity and 3'-5' exonuclease activity, the main function of 3'-5' exonuclease activity is correction function, when the added core When the nucleotide is not complementary to the temp...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12N15/10C12R1/19
CPCC12N9/1252C12N15/70C12Q1/686C12Y207/07007C12N2800/101C12Q2521/101C12Q2527/125Y02A50/30
Inventor 朱升龙王振张靖伟叶贤龙陈永泉
Owner JIANGNAN UNIV