Preparation method and application of rapid DNA ligase

A technology of DNA ligase and enzyme activity, which is applied in the field of genetic engineering, can solve the problems of low connection efficiency, time-consuming and labor-intensive gene cloning, and low DNA connection efficiency, and achieve the effect of shortening time and high gene cloning efficiency

Pending Publication Date: 2020-06-05
苏州博睐恒生物科技有限公司
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Problems solved by technology

The difference between this reaction temperature and the optimal reaction temperature of T4 DNA ligase is too large, which leads to low efficiency of DNA ligation and requires a long reaction time of up to 12 hours, making gene cloning time-consuming and laborious.
For gene cloning of blunt-ended target DNA, the connection efficiency is lower because there is no complementary base between the target gene and the vector

Method used

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  • Preparation method and application of rapid DNA ligase
  • Preparation method and application of rapid DNA ligase
  • Preparation method and application of rapid DNA ligase

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Preparation of Rapid DNA Ligase from Psychrophilic Bacteria Colwellia psychrerythraea

[0030] The first step is to design and synthesize the DNA ligase gene of the psychrophilic bacterium Colwellia psychrerythraea, and insert it between the NdeI and BamHI sites of the pET28 expression vector, fuse the archaeal histone and DNA ligase at the C-terminus, and insert 12 Alanine, construction of a recombinant expression plasmid for fast DNA ligase from the psychrophilic bacterium Colwellia psychrerythraea. The N-terminal of the fast DNA ligase has 6 consecutive histidine affinity purification tags derived from the pET28 vector, which is used for immobilized nickel ion affinity chromatography purification.

[0031] In the second step, the fast DNA ligase of the psychrophilic bacterium Colwellia psychrerythraea was recombinantly expressed. The rapid DNA ligase recombinant expression plasmid of the psychrophilic bacterium Colwellia psychrerythraea was transformed int...

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Abstract

The invention discloses a preparation method and application of a rapid DNA ligase based on a low-temperature bacterial DNA ligase. The ligation product of the rapid DNA ligase prepared by the invention after reacting for 5 minutes in a range of 4-20 DEG C is greater than 90%, and meanwhile, the DNA ligase is fused with a DNA binding protein, so that the DNA ligation reaction speed is increased, and the rapid DNA ligase is very suitable for ligation between a target gene and a vector in gene cloning. The rapid DNA ligase has the beneficial effects that one end of the low-temperature DNA ligaseis fused with one DNA binding protein, so that the binding speed and efficiency of the DNA ligase and a target DNA substrate are greatly improved, the DNA ligation reaction efficiency is improved, the reaction time is shortened, and the rapid DNA ligase is very suitable for low-temperature rapid DNA ligation reactions.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and application of a fast DNA ligase. Background technique [0002] The emergence of restriction endonuclease and DNA ligase has promoted the in vitro recombination technology of DNA, that is, gene cloning technology. Gene cloning technology is the basis of modern molecular biology and genetic engineering, which greatly promotes the application of protein recombinant expression and engineering strain transformation. DNA ligase is the basis of gene cloning technology, which is used to perform DNA ligation reaction between target gene DNA and carrier DNA to form circular recombinant DNA molecules. The catalytic efficiency of DNA ligase is crucial to the success of gene cloning. The DNA ligase currently used in gene cloning is T4 phage DNA ligase. The optimal temperature of T4 phage DNA ligase is 25-40 degrees. In order to promote the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/00C12N15/66C12R1/19
CPCC12N15/70C12N9/93C12Y605/01C12N15/66
Inventor 袁慧田文齐
Owner 苏州博睐恒生物科技有限公司
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