Barnes' protocol for long PCR to include a reverse-transcription step and was optimized for use with hepatitis C virus genomes as template. Starting with a very small amount of virus, we have successfully produced near full-length amplicons from a series of very different viruses including hepatitis A,B,C,E, and G. Once purified, these cDNA molecules proved to be good substrates for a variety of enzymatic reactions including sequencing. This methodology should therefore be very useful for genetic studies of these viruses and should be applicable to other viruses, even when available only at low titer. Most importantly, we have shown the feasibility of generating in one round of RT-PCR a full length cDNA of a viral genome, from which infectious RNA can be directly transcribed.
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