1. A novel strategy for one-step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus
- Author
-
Brenda G. Hunter, I.T.D. Petty, and Andrew O. Jackson
- Subjects
Genetics ,Barley stripe mosaic virus ,Base Sequence ,cDNA library ,Genetic Vectors ,Molecular Sequence Data ,Hordeivirus ,Nucleic acid sequence ,RNA ,Hordeum ,General Medicine ,DNA ,Biology ,Molecular cloning ,biology.organism_classification ,Molecular biology ,Plasmid ,Oligodeoxyribonucleotides ,Mosaic Viruses ,Complementary DNA ,Cloning, Molecular ,Plasmids - Abstract
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.
- Published
- 1988