Site-directed Mutagenesis by Novel Inverse PCR Strategy

Site-directed mutagenesis plays important roles to study protein structure-function relationship and the researchers are always confronted with the problem on how to generate a point mutation of a target gene efficiently. A novel strategy to produce a point mutation was depicted based on inverse PCR with PIAS3 as an example. Firstly, the entire PIAS3 coding sequence was amplified from MCF-7 cDNA and then cloned into the expression vector pXJ40-myc. Two sets of PIAS3 primers with specific mutation sequences were synthesized and then phosphorylated at their 5' terminus. Inverse PCR was performed with the phosphorylated primers as well as with the plasmid pXJ40myc-PIAS3 as the template. Further, the PCR products were subjected to Dpn I treatment, agarose gel purification, self-ligation and transformation into DH5 alpha. Three colonies were randomly selected for DNA sequencing. The expression of both the PIAS3 wide type and the point mutants( PIAS3 K110R and K411, 412R) was analyzed by transfection of these plasmids into 293T cells. The result showed that the PIAS3 K110R and K411,412R point mutants were successfully constructed and expressed in mammalian cells, which suggested that the novel inverse PCR stratagy can be applied to construct the point mutants of a target gene efficiently and conveniently.