Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.

摘要: In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, examined efficacy RLF on high-throughput multi-plasmid cloning. This utilizes precise DNA repair recombination systems within Escherichia coli, which allows bypass in vitro restriction ligation enzyme reactions commonly included routine cloning procedures. A homologous arm is linked 5'-end forward primer used amplify both target vector. different reverse primer. Therefore, genes can be cloned into vectors by after co-transformation amplified linearized vector, bear either end. More than twenty-four were generated method, uses two simple polymerase chain reaction steps. highly efficient any interest vector at site without sequence constraints, as no are required.