OBJECTIVETo construct a reverse transcriptase-polymerase chain reaction (RT-PCR)approach that can improve the specificity of primers while dropping down the nonspecific amplification.

METHODSIn the recent study we reported a new RT-PCR assay which improved markedly the specificity. However its efficiency of regressing nonspecific amplification remains to be accurately checked and further documented. In primer design, we looked over again some sequences that showed differences at 5' or 3' ends between human CAV1 and mouse Cav1 genes. cDNAs and the diluted plasmids which harbored the sequence of human CAV1 or mouse Cav1 gene were chosen as the templates. The ordinary PCR compared with one, of which primers modified by phosphorothioate and combined with proofreading polymerase, for their efficiencies of nonspecific amplification inhibited.

RESULTSTaq DNA polymerase without proofreading activity could efficiently catalyze the extension of primers with a single or multiple mismatched base pairs at the 3' terminus, but the kind of primer extension can be effectively blocked by phosphorothioate modified primers combined with proofreading polymerase. Compared with ordinary PCR reaction, this new PCR method can effectively regress the primer mismatched amplification of 50 ng DNA almost equaling to 2 x 10(4) unmatched template copies in a final volume of 50 microL.

CONCLUSIONCompared with the first generation of polymerases with or without proofreading activities mediating RT-PCR reaction, the introduction of nuclease-resistant 3' modified primers (3' phosphorothioate primer extension) can offer more simplicity, accuracy, and also decrease cost.