Ligase independent gene cloning process based on thio-modification
A technology of sulfomodification and cloning method, which is applied in the field of genetic engineering, can solve the problems of difficult target gene cloning and base sequence design, and achieve the effects of reducing the frequency of mutation introduction, simple cloning throughput, and high fidelity
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[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 inventive method such as figure 1 As shown, first, a phosphodiester bond between the 11th and 21st positions of the 5' end is used to amplify the target gene and the plasmid vector respectively, and a phosphodiester bond between the 11th and 21st positions of the 5' end is obtained. The ester bond is the DNA fragment of the thio-modified target gene and the linearized plasmid vector; then the prepared DNA molecules are digested with 5' exonuclease. The presence of thio-modification makes the 5' exonuclease digest the DNA Terminated at the sulfur modification, resulting in a 10-20 nucleotide long 3'single-stranded overhang; since the target gene is complementary to the 3'single-stranded overhang of the linearized plasmid vector, the hybridization of the two will form a Recombination...
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Abstract
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