Novel strategy of gene construction by simultaneous phosphorylation and ligation

Chemical synthesis of DNA sequences is the powerful tool in the studies of gene structure, modification, expression and function. Gene construction and consequently protein/enzymes can bridge genomics and proteomics research or facilitate commercial applications of gene and protein technologies. There is a need for simple, reproducible, less error-prone and cost-effective methods that guarantee successful synthesis of the desired genes. In order to satisfy these technical needs, we demonstrate a novel concept of gene construction usingsimultaneous reaction of phosphorylation and LCR. Combination of phosphorylation and LCR can successfully eliminate the unwanted deletion and substitution. Our method can enhance total ligation ratio by an increase of reaction temperature and removal of unphosphorylated oligonucleotides. Total process time can be shorten and the final success rate is increased successfully. Therefore, these results suggested that our gene synthesis method using simultaneous phosphorylation and ligation has much more beneficial compared to conventional gene synthesis methods.