Tagging of viral RNA transcripts with strain-specific oligonucleotides: characterization and application

Abstract

Binding(‘tagging’) of a virus-specific oligonucleotide ‘sticker’ to RNA transcripts copied from PCR products caused retardation of transcript mobility in gel. This enables detection of specific sequences within the RNA transcripts, and a virus strain (PVY NTN) could thus be positively identified. We have demonstrated further that oligonucleotides that contained virus sequences originated from different genomic locations varied in their inhibitory effect on the rate of transcript migration in gel; thus, the most effective oligonucleotide could be chosen. Combinations of different strain-specific oligonucleotides had additive retarding effects on transcript migration. The conditions for annealing oligonucleotides to the RNA transcripts were studied, including concentrations of oligonucleotides and salt. A higher electrophoresis temperature (up to 45 °C) reduced the gel retardation phenomena, which indicated a conformation mechanism. The applicability of ‘tagging’ of RNA transcripts with a strain-specific oligonucleotide for virus strain differentiation is discussed.