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DNA polymerase, coding gene thereof, preparation method for DNA polymerase and PCR application of DNA polymerase

A polymerase and gene technology, applied in the biological field, can solve the problems of lower amplification efficiency, poor extension efficiency, and low fidelity

Inactive Publication Date: 2020-12-04
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0005] At present, commercial DNA polymerases still have problems such as low thermal stability, low fidelity, poor extension efficiency, and the amplification efficiency will be reduced in the presence of pollutants or inhibitors, which cannot fully meet the needs of scientific research.

Method used

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  • DNA polymerase, coding gene thereof, preparation method for DNA polymerase and PCR application of DNA polymerase
  • DNA polymerase, coding gene thereof, preparation method for DNA polymerase and PCR application of DNA polymerase
  • DNA polymerase, coding gene thereof, preparation method for DNA polymerase and PCR application of DNA polymerase

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Embodiment Construction

[0023] Preparation of DNA polymerase encoded by Thermococcus eurythermalis and its application in PCR

[0024] 1. Construction of prokaryotic recombinant expression vector

[0025] Using the genome sequence of Thermococcus eurythermalis obtained by sequencing, the DNA polymerase gene encoded by it was determined according to bioinformatics analysis, and the putative Thermococcus eurythermalis heat-resistant DNA polymerase (PolB) sequence was obtained, as shown in sequence 1 in the sequence listing.

[0026] Firstly, using Thermococcus eurythermalis genomic DNA as a template, the putative PolB gene was amplified using the primer pair F1 and R1. The agarose gel electrophoresis results of PCR amplification of the full-length polB gene encoded by Thermococcus eurythermalis are as follows: figure 1 As shown, a 3939bp PolB gene was obtained.

[0027] The primer sequences are as follows:

[0028] F1:5'-TGCCGCGCGGCAGCCATATGATTCTCGATACCGACTAC-3'

[0029] R1: 5'-TCGAGTGCGGCCGCAAGCTT...

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Abstract

The invention provides a DNA polymerase, a coding gene thereof, a preparation method for the DNA polymerase and PCR application of the DNA polymerase. The DNA polymerase is derived from a thermophilicmicroorganism, i.e., Thermococcus eurythermalis, has the characteristics of high thermal stability, high amplification capability, high DNA amplification yield and high amplification fidelity and canbe applied to PCR amplification of target DNA. The DNA polymerase is prepared through induced expression of a recombinant expression vector in Escherichia coli, affinity purification of immobilization nickel ions and purification of cation exchange resin. A PCR buffer solution of the polymerase is composed of 50mM of Tris-HCl (pH 8.0), 2mM of MgCl2, 50mM of KCl, 2mM of (NH4)2SO4, 0.01% of TritonX-100 and 0.005% of BSA. The DNA polymerase can be applied to amplification of a target DNA fragment reaching a length of 6kb.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA polymerase and its coding gene, a preparation method and PCR application. Background technique [0002] As the core component of DNA replicators, DNA polymerase is a key enzyme in the process of genetic information replication, processing and distribution, and exists in all life forms in the three kingdoms of bacteria, archaea and eukaryotes. The main function of DNA polymerase is DNA replication. It assembles nucleotides and synthesizes new complementary DNA to existing DNA; in addition, DNA polymerase proofreads (3'-5' exonuclease activity) and repairs (5'-3' exonuclease activity) the new strand activity) to ensure the fidelity and accuracy of the replication process. [0003] There are many kinds of polymerases, each with its own characteristics in structure and function. Among them, heat-resistant DNA polymerase is mostly used in PCR (polymerase chain reaction) technology,...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N1/21C12N15/70C12P19/34C12Q1/686
CPCC12N9/1252C12Y207/07007C12N15/70C12P19/34C12Q1/686C12Q2521/101
Inventor 刘喜朋翁妍
Owner SHANGHAI JIAO TONG UNIV
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