Nested Inverse Polymerase Chain Reactions: An Effective Method for Cloning of Full-Length Sequences of Chalcone Synthase

Chalcone synthase is the key enzyme in biosynthesis of flavonoids, which play roles in pigmentation of flowers and protection against ultraviolet and pathogens. Inverse polymerase chain reaction (IPCR) is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. In this study, IPCR united with nested PCR was successfully applied in cloning full-length sequences of three Phalaenopsis chalcone synthase genes (phchs3, phchs4, and phchs5, respectively). Firstly, routine PCR with homologous primers were performed, and gene fragments of phchs3 (1 kb), phchs4 (1.2 kb), and phchs5 (800 bp) were obtained and then sequenced. Then, inverse PCR were carried out for cloning full-length sequence of each gene. Because products were not unique in single round inverse PCR, nested PCR were performed, and the specificity was much enhanced. At last, full-length sequences of 2,499 bp for phchs3, 2,502 bp for phchs4, and 1,855 bp for phchs5 were obtained. This study proved that IPCR could be more efficient if being united with nested PCR.