Iodine dissociation type gene cloning method based on sulfo-modifiers

Abstract

The invention discloses an iodine dissociation type gene cloning method based on sulfo-modifiers. According to the method, phosphodiester bonds between the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends of primers of a target gene and a plasmid vector are amplified into the sulfo-modifiers; a base sequence at the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends of forward primers of the target gene and the plasmid vector and a base sequence at the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends of reverse primers of the target gene and the plasmid vector are matched in a supplementary mode; after the target gene and the plasmid vector are subjected to PCR amplification, a PCR product is purified; iodine selectively breaks the phosphodiester bonds of the sulfo-modifiers, and under the combined action of existence of the sulfo-modifiers at the positions of 1-15, 16, 17, 18, 19 or 20 of the 5' ends and iodine heat treatment, DNA will generate a 3' single-link protruding end where 20 nucleotides grow. Through the method, cloning of the target gene does not rely on restriction endonuclease, DNA ligase or any other DNA modification enzyme, operation flux is large, and the method can be applied to large-scale gene cloning.

Description

technical field [0001] The invention relates to a new gene cloning method, in particular to a gene cloning method based on sulfomodification and iodine cleavage, which can be used for expression vector construction and gene point mutation, 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 first uses restriction endonuclease to digest the DNA fragment of the target gene and the plasmid vector, and then DNA ligase connects and circularizes the target gene and the plasmid to form a complete recombinant plasmid. Due to the low efficiency of the restriction endonuclease digestion reaction and the DNA ligase reaction, especially the restriction endonuclease digestion reaction, the percentage of positive recombinant plasmids containing the target gene is often lower than 10...

AI Technical Summary

Problems solved by technology

Compared with the chemical method, because the enzyme is a protein, it is relatively unstable, and has the same disadvantages as other enzymatic gene cloning techniques, that is, the cloning efficiency is unstable

At the same time, the thio modification in the patent 200710039383 can only block the digestion of exonuclease to a certain extent, and cannot completely block the nuclease digestion reaction at the thio modification site, which will still cause a large degree of excessive DNA Digestion, reducing cloning efficiency

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