Application and seamless cloning method of DNA exonuclease

Abstract

The invention provides application of DNA exonuclease to DNA recombinant seamless cloning and provides a kit capable of being used for DNA recombinant seamless cloning. The DNA exonuclease is T5 exonuclease, T7 exonuclease, exonuclease III or Lambda exonuclease or a mixture thereof. The invention further provides a seamless cloning method applying the DNA exonuclease, which comprises the followingsteps: linearizing a vector through a PCR method or restriction endonuclease digestion; introducing homologous sequences that are respectively homologous to the two ends of the vector at the two endsof a target gene fragment through PCR to obtain an amplified target gene fragment; after the treated target fragment and the treated linearized vector are mixed, adding the DNA exonuclease and a reaction solution for temperature bath to obtain a vector and fragment mixture; converting an echerichia coli receptive cell by using the obtained vector and fragment mixture. The application and the method provided by the invention have the following advantages that the site selection can be flexibly performed, and the gene cloning can be performed in any position of the vector; the vector construction can be quickly, simply and conveniently completed within 10 minutes; meanwhile, the cloning is accurate and efficient.

Description

technical field [0001] The present invention relates to molecular biology, in particular to a seamless cloning method of DNA recombination. Background technique [0002] In the 1970s, the Boyer-Cohen experiment successfully constructed a double-drug-resistant recombinant plasmid, the prelude to genetic engineering, and then O.Smith discovered the first restriction endonuclease. So far, the most traditional is also The most classic gene cloning technology - Ligation-dependent cloning technology was born. The ligation-dependent cloning technique uses restriction endonucleases to treat the vector and PCR product fragments to produce the same cohesive ends, and then ligates into a circle under the action of DNA ligase to transform the host cells. As one of the most important technologies in the field of biology, gene cloning technology has always had huge market potential and prospects. Many well-known enterprises and companies such as NEB, Novagen, Thermo scientific, etc. have...

AI Technical Summary

Problems solved by technology

Therefore, the shortcoming of this kind gene cloning technology method always exists: (1) the two ends of carrier and object fragment will have identical restriction enzyme cutting site; (2) the recovery efficiency after vector and fragment digestion is too low; ( 3) Easy self-ligation, high false positive; (4) Subcloning is required if ligated to T vector; (5) Insertion direction needs to be identified during blunt-end ligation, which has not been effectively resolved

However, this technology mainly relies on recombinant enzymes. There are many types of recombinant enzymes on the market, and the quality is also uneven. Some products have recombinant enzymes that are mixed enzymes, which undoubtedly increases the cost.

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