Improved yield of full-length phaseolin cDNA clones by controlling premature anticomplementary DNA synthesis

The manner in which the known enzymatic properties of reverse transcriptase may limit the length of double stranded cDNAs to be used in cloning was studied. Results here suggest that the well-documented ability of reverse transcriptase to synthesize anticomplementary DNA can, if unrecognized, seriously limit the final yield of full-length cDNA clones. Under conditions which permitted anticomplementary DNA synthesis during the synthesis of the first cDNA strand, no full-length cDNA clones for phaseolin, the principal storage proteins ofPhaseolus vulgaris, were detected among 19 phaseolin-positive cDNA clones. When anticomplementary DNA synthesis was inhibited with 4 mM sodium pyrophosphate, 5 full-length cDNA clones were identified among 45 phaseolin-positive clones. In both cases, the products of the first strand synthesis were C-tailed and the second strand synthesized by reverse transcriptase using oligo(dG) as a primer. The implications of anticomplementary synthesis in cloning methods involving the use of S1 nuclease are discussed. In addition, a rapid, one-step procedure for obtaining partial clones which equally represent the 5' and 3' ends of the RNA is presented.