OBJECTIVETo introduce a new technique for rapid construction of gene site-directed mutagenesis.

METHODSThree primers are synthesized. One is a primer with the needed mutation; the other two containing appropriate enzyme sites for construction of the PCR fragment into a suitable plasmid are located at the flanks of the mutation primer. After the amplification of the PCR fragment using the mutation primer and the reverse flanking primer, another PCR is performed using the previous PCR mutation segment as primer and the other flanking primer. The final PCR segment can be cloned into an appropriate plasmid by using the enzyme sites in the primers.

RESULTSTwo site-directed mutagenesis have been successfully constructed in the Parkin gene by this method.

CONCLUSIONThe method is effective and simple for construction of gene site-directed mutagenesis.