RAPID CLONING OF UNCHARACTERIZED TOBACCO RATTLE VIRUS ISOLATES USING LONG TEMPLATE (LT) PCR

A rapid method was developed using long template (LT)-PCR to amplify t he complete RNA2 of isolates of TRV for which no sequence data are ava ilable. The amplification makes use of a 5' terminal oligonucleotide w hich contains degeneracies corresponding to the sequences of several d ifferent TRV isolates, and a 3' oligonucleotide which is complementary to a sequence present in all known isolates. This method was used to show the high degree of sequence homology existing in the terminal reg ions of two uncharacterised TRV isolates (TPO3 and PAY4), and revealed the deletion of an 80-nucleotide sequence in the 5' terminal region o f TPO3 RNA2.