A METHOD FOR ACCURATE DETERMINATION OF TERMINAL SEQUENCES OF VIRAL GENOMIC RNA

A combination of ligation-anchored FCR and anchored cDNA cloning techn iques were used to clone the termini of the saguaro cactus virus (SCV) RNA genome. The terminal sequences of the viral genome were subsequen tly determined from the clones. The 5' terminus was cloned by ligation -anchored PCR, whereas the 3' terminus was obtained by a technique we term anchored cDNA cloning. In anchored cDNA cloning, an anchor oligon ucleotide was prepared by phosphorylation at the 5' end, followed by a ddition of a dideoxynucleotide at the 3' end to block the free hydroxy l group. The 5' end of the anchor was subsequently ligated to the 3' e nd of SCV RNA. The anchor-ligated, chimerical viral RNA was then rever se-transcribed into cDNA using a primer complementary to the anchor. T he cDNA containing the complete 3'-terminal sequence was converted int o ds-cDNA, cloned, and sequenced. Two restriction sites, one within th e viral sequence and one within the primer sequence, were used to faci litate cloning. The combination of these techniques proved to be an ea sy and accurate way to determine the terminal sequences of SCV RNA gen ome and should be applicable to any other RNA molecules with unknown t erminal sequences.