A novel strategy for one-step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus.

We have devised a novel vector-primer strategy for cloning of full-length (FL) cDNA which can be applied to non-polyadenylated RNA species. Single-stranded plasmid DNA is used to prime first-strand synthesis by reverse transcriptase, and plasmids covalently linked to FL cDNA are then circularized by the annealing of a specific oligodeoxyribonucleotide (band-aid oligo). Only limited nucleotide sequence data are required from the termini of each RNA species to be cloned to design the plasmid primer and band-aid oligo. The band-aid strategy has been applied to the cloning of barley stripe mosaic virus genomic RNAs, and found to be both rapid and efficient. A strategy for the preparation of linear double-stranded plasmid DNA templates (suitable for run-off in vitro transcription) which is independent of restriction sites present within the cloned cDNA is also described.