Efficient connection method of DNA fragment and carrier and application thereof, and kit

Abstract

The invention discloses an efficient connection method of a DNA fragment and a carrier. The method comprises the first step of an enzyme-digested and connection reaction, wherein the carrier is mixed with the DNA fragment, and a buffer solution, restriction endonuclease and DNA ligase are added into the mixture to be subjected to the enzyme-digested and connection reaction; the second step of an enzyme-digested and dephosphorylation reaction, wherein restriction endonuclease and alkaline phosphatase are added into the system after the reaction is over, and then the buffer solution is replenished to be subjected to the enzyme-digested and dephosphorylation reaction. According to the efficient connection method of the DNA fragment and the carrier, the carrier does not need to be subjected to linearization preparation in advance, and thus the enzyme-digested reaction can be achieved while the connection is conducted; meanwhile, after a reaction product is converted, only white spots grow on a culture dish and the positive clone efficiency is obviously improved, which facilitates subsequent selection and culture of bacterial colonies and PCR identification of bacteria liquid; meanwhile, the workload of selection and culture of bacterial colonies and PCR identification of bacteria liquid in large-scale gene synthesis is reduced, the cost of gene synthesis and clone is reduced, and thus the efficient connection method of the DNA fragment and the carrier is particularly applicable to large-sample sequencing analysis of target genes.

Description

technical field [0001] The present invention relates to DNA assembly technology, in particular to an efficient connection method of double-stranded DNA molecule fragments and cloning vectors. Background technique [0002] In genetic engineering and protein engineering, the research on the function of the target gene is an important direction: first, to determine its expression regulation mechanism and biological function; second, to establish a high-efficiency expression system for in vitro gene function expression, such as antibody engineering; The target gene undergoes necessary structural and functional modifications in vitro, including human gene therapy; or some other aspects. In these studies, it is often necessary to obtain a large number of target genes, and gene cloning and recombination is an important basic technology to achieve this research purpose. The linking of target gene DNA fragments and cloning vectors is one of the important links in gene cloning and ...

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Problems solved by technology

[0005] Aiming at the deficiencies of the existing technology and the actual needs in the process of scientific research and development, the present invention provides a method for efficiently linking DNA molecular fragments and carriers, which can solve the problem of low connection efficiency of blunt-end cloning in the process of blunt-end ligation, and after transformation coating culture There are many blue spots and few white spots on the petri dish, and there are false positive clones. The workload of subsequent colony selection and culture identification is heavy, it is difficult to pick white spots, double clones are prone to occur, and it is not suitable for large sample sequencing analysis.

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