A kind of high-fidelity dna polymerase and its preparation and application

A polymerase and high-fidelity technology, which is applied in the field of genetic engineering, can solve the problems of low reaction sensitivity, limit the use and promotion of PfuDNA polymerase, and low amplification efficiency, and achieve the effects of improving sensitivity, high fidelity, and improving amplification efficiency

Active Publication Date: 2017-09-01
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, Pfu DNA polymerase is generally used as the preferred high-fidelity DNA polymerase for high-fidelity amplification of nucleic acids. However, in practical applications, Pfu DNA polymerase has relatively high requirements on the structure, purity and concentration of primers and samples. , the reaction sensitivity is low, and the amplification efficiency is low, which limits the use and promotion of Pfu DNA polymerase

Method used

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  • A kind of high-fidelity dna polymerase and its preparation and application
  • A kind of high-fidelity dna polymerase and its preparation and application
  • A kind of high-fidelity dna polymerase and its preparation and application

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Embodiment 1

[0060] The acquisition of the DNA molecule of embodiment 1 coding Tco DNA polymerase

[0061] The bacterial strain Thermococcus coalescens was purchased from the Japan Collection of Microorganisms, and its preservation number is JCM:12540. Centrifuge the purchased bacterial species at 12000rpm for 5min, discard the supernatant; for the cell pellet, refer to Promega’s Genomic DNA Purification kit extracts genomic DNA, the brief steps are as follows:

[0062] Cell pellets were resuspended in 600 μL Nuclei Lysis Solution, and the cells were lysed in a water bath at 80°C for 5 minutes; after cooling to room temperature, 200 μL Protein Precipitation Solution was added, shaken and mixed, and placed in an ice bath for 5 minutes; centrifuged at 12,000 rpm for 5 minutes; pipette the supernatant into another clean centrifuge tube , add 600 μL of isopropanol, mix well and ice-bath for 10 minutes; centrifuge at 12,000 rpm for 5 minutes; wash the nucleic acid pellet twice with 700 μL of...

Embodiment 2

[0084] Embodiment 2 Contains the construction of the recombinant vector of the DNA molecule of coding Tco DNA polymerase

[0085] The target fragment of about 2.3 kb in full length obtained in Example 1 was digested with BamH I and Not I, ligated into the same digested vector containing T7 promoter, and transformed into DH5α competent cells.

[0086] Confirm the correctness of the gene sequence encoding Tco DNA polymerase through bacterial liquid PCR identification, enzyme digestion identification and sequencing verification, culture and verify the correct competent cells, and extract the plasmid, the obtained plasmid is the DNA polymerase encoding Tco Recombinant Vector of DNA Molecule.

Embodiment 3

[0087] The preparation of the transformant that embodiment 3 expresses Tco DNA polymerase fusion protein

[0088] Transform the recombinant vector containing the DNA molecule encoding Tco DNA polymerase obtained in Example 2 into the host cell E.coli Rosetta (DE3), pick a single colony and culture it to OD 600 = 0.4, add IPTG to a concentration of 0.1mmol / L for induction for 4h, collect the cells and ultrasonically disrupt them, detect the expression of the target protein by SDS-PAGE electrophoresis, and find that the prepared transformant can express Tco DNA polymerase fusion protein.

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Abstract

Provided are a high-fidelity DNA polymerase, a gene for coding the high-fidelity DNA polymerase, a method for preparing the high-fidelity DNA polymerase, and a use thereof in nucleic acid amplification. The high-fidelity DNA polymerase has an amino acid sequence represented by SEQ ID NO:1, can be used to amplify a nucleic acid template with a high GC content or a nucleic acid template with a low sample purity, and can amplify a very low template amount.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a high-fidelity DNA polymerase and its preparation and application. Background technique [0002] DNA polymerase is widely used in molecular biology research, such as DNA sequencing, labeling, mutation and nucleic acid amplification reactions. Due to its thermal stability, thermostable DNA polymerase will not be inactivated under the condition of high temperature denaturation of nucleic acid, and is currently a commonly used enzyme for nucleic acid amplification. According to the difference in sequence, DNA polymerase can be divided into six major families: A, B, C, D, X and Y. Thermostable DNA polymerase mainly belongs to family A and family B, and has similar biological properties. The representative of family A DNA polymerase is Taq DNA polymerase, which is isolated from the genome of Thermus aquaticus YT-1, has 5'-3'DNA exonuclease activity and 5'-3'DNA polymerase activity, i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N15/10
CPCC12N9/1252C12Y207/07007C12N9/1241
Inventor 葛猛夏霞宇余倩王宏伟
Owner 北京旌准医疗科技有限公司
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