Purification of full-length enterovirus cDNA by solid phase hybridization capture facilitates amplification of complete genomes

The aim of the study was to develop a method for the selective purification of full-length enterovirus single strand (ss) cDNA for subsequent amplific ation of complete enterovirus genomes by long distance PCR. As a model syst em we have used the prototype strain of echovirus 5 (EV5). Due to inefficie nt first strand cDNA synthesis using EV5 RNA as template, only a few molecu les of EV5 sscDNA were completely reverse transcribed and no amplification products were observed when long distance polymerase chain reaction (LD-PCR ) was used for amplification of complete EV5 genomes. To purify the complet e EV5 cDNA present, an oligonucleotide, derived from the conserved 5' end o f an enterovirus genome, was immobilized on paramagnetic beads and complete EV5 sscDNA was captured and purified from the less than full-length cDNAs. LD-PCR using the purified EV5 cDNA resulted in amplification of complete E V5 genomes. Transfection of the EV5 RNA transcribed from these uncloned amp licons resulted in production of replicating viruses. This demonstrates tha t solid phase hybridization capture of sscDNA is an efficient method that c an be used for enrichment and purification of full-length enterovirus sscDN As. (C) 1999 Elsevier Science B.V. All rights reserved.