Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)-T7 primed cDNA.

products were examined by Southern hybridization with internal oligonucleotide probes and by sequence analysis (not shown). In the study reported here, the ability of the reverse transcriptase enzyme to transcribe full-length cDNA from RNA was followed by a Long PCR reaction to amplify the entire enteroviral coding region together with the 5' and 3' nontranslated regions. The method was tested on CBV2 virus cultured in HEp2 cells (Figure I), but has now been employed for other enteroviruses, including polioviruses 1 and 3, coxsach e A16 and echovirus 9 (not shown). This novel application of the PCR technique has a number of advantages over previous PCR methods for picornaviruses. It will now be possible to rapidly generate full-length infectious clones of viral isolates for genetic analysis and site-directed mutagenesis. Previous attempts at isolating fulllength cDNA clones depended on the screening of a multitude of clones; for instance, Kandolf and Hofschneider (5) screened 4800 cDNA clones before isolating a full-length CBV3 clone.