The use of PCR for cloning of large cDNA fragments of turnip mosaic potyvirus.

A method is described whereby turnip mosaic virus RNA (TuMV RNA) was reverse transcribed and the resulting cDNA amplified enzymatically using the Taq DNA polymerase and degenerate oligonucleotide primers. Two degenerate oligonucleotide primers based on regions of homology in the amino acid sequence of the cytoplasmic inclusion protein and the nuclear inclusion b protein from five potyviruses were synthesized. Polymerase chain reactions utilizing these degenerate primers in association with specific primers amplified a 1.2 kb and a 3.3 kb fragment. These amplified fragments were dC-tailed and cloned into pUC9. Their partial sequence, when compared to potyvirus sequences, showed that they were derived from TuMV RNA and approximately 4.4 kb of viral genome was cloned.