Long RT-PCR Cloning — Amplification of Full-Length Enterovirus Genomes

Abstract

At the present time, there is widespread interest in the use of polymerase chain reaction (PCR) amplification techniques to generate DNA or cDNA sequences for target identification, sequence determination, subsequent use as molecular probes, or as functional units which can be expressed in appropriate vector systems. This chapter describes the technique of Long RT-PCR followed by cloning of the PCR products into plasmid vectors. Essentially, this is a method for copying RNA into first-strand cDNA using the enzyme reverse transcriptase (RT) and then PCR amplifying the cDNA to give rise to a double stranded cDNA copy of the original RNA target sequence. The basic technique has been taken to a more advanced stage by the introduction of reverse transcriptase enzymes such as SUPERSCRIPT II RNase H^- (Invitrogen Liefe Technologies Ltd) which allow longer stretches of RNA to be copied into DNA and Taq polymerases such as rTth DNA polymerase, XL (PE Applied Biosystems), Deep Vent DNA polymerase (New England Biolabs) and the polymerase mix provided with the Advantage 2 PCR Enzyme System (BD Biosciences, Clonetech, UK) which have high fidelity rates and can generate much longer PCR products. By incorporating restriction enzyme sites at the ends of the amplification primers, it is possible to clone these long PCR cDNA fragments into suitable cloning sites on a large number of commercial vectors.