A Cool DNA Cloning And Assembling Method Based On T5 Exonuclease

Gene cloning has become one of the most commonly used techniques in scientific research.A limiting step in this process is the directional insertion of the target DNA into the cloning vector using restriction enzymes and DNA ligases.This procedure suffers from several limitations,including the lack of tight junctions and the provision of unique restriction sites in the inserted fragments and vectors.With the emergence and development of metabolic engineering and synthetic biology,the requirements for cloning methods are getting higher and higher.Some DNA cloning techniques that do not rely on ligation have overcome the restriction of restriction sites,but they are time-consuming and costly.Recently,cloning methods based on homologous recombination have become more and more popular.In this study,we proposed a Cool Cloning assembly method based on T5 exonuclease.By primer design and PCR amplification,the two ends of the inserted fragment were attached with homologous ends of the two ends of the linear vector.In the system containing T5 exonuclease,ice bath for 5 min,using the 5 '-3 ' exonuclease activity of T5 exonuclease,the linear plasmid carrier and the inserted fragment were used to form the circular DNA molecule with single chain notch,which was then transferred into competent E.coli cells to complete the defect repair and covalent connection by means of the intracellular repair system.The method can also be used to assemble multiple DNA fragments at the same time.The reaction mixture is simple and 0.5 U of T5 exonuclease is used for each reaction.In this paper,the enzyme concentration,reaction time,reaction temperature,the amount of DNA,homologous arm length and other conditions have been optimized,the efficiency of 2 fragments assembly can reach more than 95%.The research of Cool Cloning proposed in this study has the following characteristics:(1)T5 exonuclease was used to make the carrier and insert fragments produce long complementary viscous ends and improve the recombination efficiency.(2)After 5 minutes of Cool Cloning ice bath reaction,it can be directly transformed into E.coli cells,which is simple to operate and easy to conduct large-scale high-throughput operation,and can quickly complete DNA molecular assembly.(3)The positive rate of the assembly was high,and more than 95% positive clones could be obtained.(4)The assembly can simultaneously complete the assembly of multiple DNA fragments.