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 amplification of complete enterovirus genomes by long distance PCR. As a model system we have used the prototype strain of echovirus 5 (EV5). Due to inefficient first strand cDNA synthesis using EV5 RNA as template, only a few molecules 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 complete EV5 cDNA present, an oligonucleotide, derived from the conserved 5' end of 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 EV5 genomes. Transfection of the EV5 RNA transcribed from these uncloned amplicons resulted in production of replicating viruses. This demonstrates that solid phase hybridization capture of sscDNA is an efficient method that can be used for enrichment and purification of full-length enterovirus sscDNAs.
Lindberg
A M
AM
Institute of Natural Sciences, University of Kalmar, Sweden. michael.lindberg@ng.hik.se
Andersson
A
A
D002460:Cell Line; D018076:DNA, Complementary / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification*; D029822:Enterovirus B, Human / Q000235:genetics*; D005784:Gene Amplification; D016679:Genome, Viral; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods
D018076:DNA, Complementary; D004279:DNA, Viral