A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs

• Published in: Analytical biochemistry Vol. 203; no. 2; pp. 269 - 273
• Main Authors: Lakshman, Dilip K., Tavantzis, Stellos M., Boucher, Alain, Singh, Rudra P.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.06.1992; Elsevier
• Subjects: Autoradiography; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA - genetics; DNA Restriction Enzymes; Electrophoresis, Agar Gel; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Molecular cloning; Plants - microbiology; RNA - genetics; RNA, Satellite; RNA, Viral - genetics; Viroids - genetics; Monocotyledones; Vegetals; Escherichia coli; Method; Virus; Plasmid; Complementary DNA; Viroid; Angiospermae; Restionaceae; Bacteria; Circular RNA; Spermatophyta; Molecular cloning; Enterobacteriaceae; Linearization
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(92)90312-U

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and β-NAD +. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5α. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.