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KOD DNA polymerase mutant, and preparation method and application thereof

A mutant and polymerase technology, applied in the field of KOD DNA polymerase mutants and their preparation, can solve the problems of inability to amplify gene fragments, reduced extension performance, etc., and achieve the expansion of application scope, improvement of inhibitor resistance, and PCR reaction performance. boosted effect

Active Publication Date: 2021-11-05
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The exo-cutting activity of the mutant H147K is 276% of that of the wild type, but its elongation performance is reduced, and a 3.6kb gene fragment cannot be amplified

Method used

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  • KOD DNA polymerase mutant, and preparation method and application thereof
  • KOD DNA polymerase mutant, and preparation method and application thereof
  • KOD DNA polymerase mutant, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 constructs the recombinant vector containing the nucleotide sequence of coding KOFU-S DNA polymerase

[0058] (1) According to the amino acid sequence (SEQ ID NO.1) of the mutant KOFU-SDNA polymerase, after codon optimization of the E. coli expression system, a DNA molecule capable of high-efficiency expression in E. coli was obtained, and splicing was used to In the PCR method, the DNA molecule encoding the mutant KOFU-SDNA polymerase is artificially synthesized, specifically as shown in SEQ ID NO:2.

[0059] (2) The DNA molecule encoding the mutant KOFU-SDNA polymerase was recombined with the expression vector pET-28a. The synthetic primer sequences are as follows:

[0060] NdeI-FP: 5'-GCGC CATATG ATGTTAACCATC-3' (SEQ ID NO: 4);

[0061] XhoI-RP: 5'-GCGC CTCGAG TTATTTTTTCTG-3' (SEQ ID NO: 5).

[0062] PCR was used to amplify the DNA molecule of the mutant KOFU-S DNA polymerase, and the artificially synthesized DNA molecule encoding the mutant KOFU-...

Embodiment 2

[0064] The preparation of the transformant of embodiment 2 expression mutant KOFU-SDNA polymerase

[0065] Transform the recombinant vector obtained in Example 1 into the host cell E.coli BL21(DE3), pick a single colony, inoculate into liquid LB medium and cultivate to OD 600 was 0.8, added IPTG to a final concentration of 0.1mmol / L, induced at 18°C ​​for 16h, collected the cells and sonicated them, and then detected the expression of the target protein by SDS-PAGE electrophoresis. It was found that the prepared transformants could highly express the mutant KOFU- SDNA polymerase.

Embodiment 3

[0066] Expression and identification of embodiment 3 mutant KOFU-SDNA polymerase

[0067] The positive transformant strains capable of expressing the mutant KOFU-SDNA polymerase obtained in Example 2 were inoculated into 60 mL LB medium containing 50 μg / mL kanamycin sulfate, and placed in a shaker at 37° C. for shaking culture overnight; Take the seed solution cultivated overnight, inoculate it into 500 mL LB medium containing 50 μg / mL kanamycin sulfate at a ratio of 1:100, and continue shaking culture in a shaker at 37 ° C for 4 h (OD 600 =0.6~0.8); IPTG was added to each bottle of bacterial solution to a final concentration of 0.1mmol / L, and the shaking induction was continued at 18°C ​​for 18 hours; the induced bacterial cells were collected by centrifugation and weighed, and the wet weight of the bacterial cells was recorded; 1 mL of each bacterial solution was centrifuged at 12,000 rpm for 1 min, and the precipitate was collected, and 150 μL of lysis buffer was added, osc...

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Abstract

The invention discloses a KOD DNA polymerase mutant, and a preparation method and application thereof, and belongs to the technical field of biology. The KOD DNA polymerase mutant KOFU-S disclosed by the invention is formed by carrying out deletion, site-specific mutagenesis, chimeric treatment and fusion of double-chain nucleic acid binding protein Sso7d on wild type KODDNA polymerase. Compared with a wild type KODDNA polymerase, the KOD DNA polymerase mutant disclosed by the invention has higher fidelity; meanwhile, the PCR amplification performances such as amplification rate, extension capacity and inhibitor resistance is improved; and the application of the polymerase in the field of molecular biology is widened.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a KOD DNA polymerase mutant and its preparation method and application. Background technique [0002] Polymerase chain reaction (PCR) is one of the most important basic research methods in modern molecular biology, and it has been recognized as an essential technique for studying gene functions. A key component in PCR technology is a thermostable DNA polymerase. [0003] Before the heat-resistant polymerase was applied, the PCR program used Escherichia coli DNA polymerase which was not heat-resistant, and the denaturation temperature in the PCR reaction could inactivate it. Therefore, in the PCR process at that time, it was necessary to Adding new polymerases during the cycle makes the operation complicated. However, thermostable DNA polymerase still has polymerase activity at 95°C, so there is no need to add polymerase in each cycle of PCR, which greatly simplifies the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21C12N15/10C12R1/19
CPCC12N9/1252C12N15/70C12Q1/686C12Y207/07007C12N2800/22C12N2800/101C12Q2527/125C12Q2521/101Y02A50/30
Inventor 胡松青何贤蓉刘光毅侯轶
Owner SOUTH CHINA UNIV OF TECH
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