A one-step, seamless, non-homologous, multi-segment gene splicing transformation method and its kit

Abstract

The invention discloses a one-step seamless, non-homologous and multi-fragment gene splicing method. The steps of DNA fragment phosphorylation, DNA fragment bridging and exponential type amplification of a ligation product are finished in one pipe through thermal cycling similar to PCR. The method has the following advantages: (1) ligation reaction is finished by one step, so that high cost caused by using a phosphorylation primer is avoided; (2) the concentration of DNA, the concentration of a bridging primer and components and a proportion of a buffer solution are optimized, and the ligation reaction with high reliability and good repeatability is reached; (3) DNA fragments are seamlessly ligated with one another without introduction of excess nucleotide; (4) non-DNA sequence dependency is realized; (5) the multi-fragment gene splicing ability is efficient; (6) the ligation product is amplified while ligation, so that the concentration of a product is greatly increased, and efficient conversion and subsequent experiment needs are guaranteed; and (7) the cost is low compared with that of the conventional restriction enzyme ligation method.

Description

technical field

[0001] The invention relates to the technical field of molecular biology, more specifically, to a one-step, seamless, non-homologous, multi-segment gene splicing and transformation method and a kit thereof. Background technique

[0002] DNA splicing technology is one of the most important technical methods in current biological research, and most of molecular biology and cell biology are based on this technology. However, since Cohen and other researchers recombined the first DNA molecule 40 years ago, the mainstream DNA manipulation method is still based on the original enzyme-cut ligation method. There are 6 bp nucleotide scars left in the generated DNA products; at the same time, this traditional method cannot meet the complex and diverse application requirements of today. For example, multi-fragment ligation in metabolic engineering, standardization and reuse of DNA fragments in synthetic biology, and efficient and reliable splicing of DNA fragments of d...

Images