Cloning independent site-directed mutagenesis using total RNA as template

Several site-directed mutagenesis methods have been developed and are being currently used in research. All these methods apply three or four prim-ers, including one or two bearing the ap-propriate mutation, and most use PCR to amplify the desired sequence and to incorporate the muta-tion (1–6). Although several developments have improved the robustness and reliabil-ity of the method, all of them require DNA clones as tem-plates. Moreover, purification of the intermediate reaction products and separation and identification of the mutat-ed sequence make some of these methods laborious and complicated. In this report we present a novel approach combining reverse transcrip-tion (RT) and PCR tech-niques, which starts from total RNA, eliminates the need of cDNA clones, and uses uni-versal primers and only one gene-specific primer.Our protocol involves two parallel RT reactions (RT1 and RT2) and one PCR am-plification (Figure 1). Both RT reactions are started from the same total RNA contain-ing the expressed target gene. An RNase H