A polymerase chain reaction method adapted for selective amplification and cloning of 3' sequences of potyviral genomes: application to dasheen mosaic virus

Abstract

'Universal' degenerate oligonucleotide primers were used to amplify cDNA sequences containing the 3' untranslated region (3' UTR) and a portion of the coat protein gene sequence of dasheen mosaic potyvirus (DMV). These primers were based on the conserved WCIEN and QMKAAA 'boxes' of the potyviral coat protein and the poly-A tail found at the 3' end of the genome. The forward genome-sense primers were designed taking into consideration the codon degeneracy of the WCIEN and QMKAAA residues for several potyviruses. The anti-sense reverse primer has 21 T residues followed by either A, C or G at the 3' end to ensure specific priming at the end of the 3' UTR and beginning of the poly-A tail. The specificity of amplification was verified using the known potyviruses (watermelon mosaic 2 and soybean mosaic viruses). To demonstrate the applicability of this method, the 3' UTR of the unsequenced DMV was amplified, cloned and sequenced. Sequence comparisons with other potyviral 3' UTRs revealed DMV to be quite distinct: nucleotide sequence similarities of only 34% to 44% were found with sequenced viruses indicating no close affinities with any other potyvirus. The potyvirus 3' sequence amplification procedure is simple and rapid, is potentially useful in developing virus specific probes and may be used to differentiate strains and species of potyviruses on the basis of the 3' UTR sequences.