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 primers, including one or two bearing the appropriate mutation, and most use PCR to amplify the desired sequence and to incorporate the mutation (1–6). Although several developments have improved the robustness and reliability of the method, all of them require DNA clones as templates. Moreover, purification of the intermediate reaction products and separation and identification of the mutated sequence make some of these methods laborious and complicated. In this report we present a novel approach combining reverse transcription (RT) and PCR techniques, which starts from total RNA, eliminates the need of cDNA clones, and uses universal primers and only one gene-specific primer. Our protocol involves two parallel RT reactions (RT1 and RT2) and one PCR amplification (Figure 1). Both RT reactions are started from the same total RNA containing the expressed target gene. An RNase Hpoint mutant reverse transcriptase is used in the reaction, because it has the characteristic of adding dC to the 3′ end of the synthesized cDNA in a templateindependent manner, which can be used to prime the 3′ end of the cDNA during RT. In our method in the two separate RT reactions, two different universal primers, both having an extra 5′-GGG-3′ stretch of their 3′ end for strand-switching, are used. The first-strand cDNA generated in the first RT reaction (RT1) contains the mutated sequence and is present at the 5′ end of the first-strand cDNA linked to the universal primer U1. The RT1 reaction utilizes the mutagenic primer and one of the universal RT primers, U1. The first-strand cDNA generated in the second RT reaction (RT2) contains the full-length and nonmutant target gene with different universal binding sites at both ends. Universal RT primers U2 and U3 are used in RT2 (Figure 1, A1). The full-length mutated target gene is produced and amplified in a one-tube two-step PCR as reported previously (5). The universal primer U1 amplifies the sense strand of the cDNA of RT1 during the first PCR step (Figure 1, A2, step 1). Having stopped the reaction, the RT2 product is added to the mixture. The sense strands carrying Cloning independent site-directed mutagenesis using total RNA as template