RT-PCR amplification and cloning of large viral sequences.

Abstract

A long reverse transcription polymerase chain reaction (LRP) protocol is described for the amplification of large RNA sequences. The amplification of near full-length hepatitis C virus (HCV) genome from serum samples is used as an example to detail each step in LRP procedure, including primer design, RNA extraction, reverse transcription, and PCR. The protocol for efficient cloning of such large amplicons is also presented. Since HCV represents a difficult template in terms of its near full-length amplification due to extensive secondary structures and low titers in clinical samples, methods described in this chapter should be applicable for other RNA viruses and cellular RNA templates.