Non-dependent ligase gene cloning process based on thio-modification

A technology of thiomodification and cloning method, applied in the field of genetic engineering, can solve the problems of difficult base sequence design and target gene cloning, and achieve the effects of simple cloning throughput, reducing the frequency of introducing mutations, and high fidelity

Inactive Publication Date: 2007-11-07
苏州博睐恒生物科技有限公司
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Problems solved by technology

The operational disadvantages of this method mainly include: 1) a specific base sequence needs to be introduced at the 5' end of the target gene and the linear plasmid vector, resulting in an artificial base sequence attached to both ends of the cloned target gene; 2) the introduced A specific base sequence is sometimes difficult to design, making it difficult to clone the target gene

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  • Non-dependent ligase gene cloning process based on thio-modification

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Embodiment Construction

[0016] The technical solutions of the present invention are described in further detail below through examples. The following examples are not intended to limit the present invention.

[0017] The method of the present invention is shown in Figure 1. Firstly, a phosphodiester bond between the 11-21 positions of the 5' end is used to amplify the target gene and the plasmid vector respectively to obtain the 11-21 positions of the 5' end. A phosphodiester bond between the positions is the DNA fragment of the thio-modified target gene and the linearized plasmid vector; then the prepared DNA molecule is digested with a 5' exonuclease, and the presence of the thio-modification makes the 5' exonuclease During the digestion of DNA, it will terminate at the thio modification, resulting in a 3' single-stranded overhang of 10-20 nucleotides in length; since the target gene is complementary to the 3' single-stranded overhang of the linearized plasmid vector, the two The mixed hybridizati...

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Abstract

The present invention relates to non-dependent ligase gene cloning process based on thio-modification. Each of the amplified target gene and the primer of the plamid vector has The 5' end thio-modified. The cloning process includes: PCR amplification of target gene and plamid vector and purification of PCR products; degrading DNA chain from the 5' end with 5' exonuclease and thio-modification to create 10-20 of 3' single chain raised ends of nucleoside; and direct transformation to colibacillus. The target gene cloning of the present invention has no dependence on restrictive endonuclease and DNA ligase and great operation flux, and may be used in large scale gene cloning.

Description

technical field [0001] The invention relates to a new gene cloning method, in particular to a ligase-independent gene cloning method based on sulfur modification, which can be used for constructing recombinant protein expression vectors and belongs to the technical field of genetic engineering. Background technique [0002] The emergence of gene cloning technology has greatly promoted the development of modern genetic engineering and protein engineering. The traditional gene cloning technology involves the restriction endonuclease digestion of the target gene DNA fragment and the plasmid vector, and the two essential operations of ligase ligation and circularization of the target gene and the plasmid. Due to the restriction endonuclease digestion reaction and the DNA ligase reaction, especially the restriction endonuclease digestion reaction, the efficiency directly determines whether the target gene cloning can be successful. Although the use of alkaline phosphomonoesteras...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 刘喜朋刘建华
Owner 苏州博睐恒生物科技有限公司
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