Rapid cloning of uncharacterised tobacco rattle virus isolates using long template (LT) PCR

A rapid method was developed using long template (LT)-PCR to amplify the complete RNA2 of isolates of TRV for which no sequence data are available. The amplification makes use of a 5' terminal oligonucleotide which contains degeneracies corresponding to the sequences of several different 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 regions of two uncharacterised TRV isolates (TPO3 and PAY4), and revealed the deletion of an 80-nucleotide sequence in the 5' terminal region of TPO3 RNA2.