title: A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs.
abstract: 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 beta-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 DH5 alpha. 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.
authors.affiliation: Department of Plant Biology and Pathology, University of Maine, Orono 04469.
authors.forename: D K
authors.initials: DK
authors.lastname: Lakshman
authors.forename: S M
authors.initials: SM
authors.lastname: Tavantzis
authors.forename: A
authors.initials: A
authors.lastname: Boucher
authors.forename: R P
authors.initials: RP
authors.lastname: Singh
mesh_terms: D001345:Autoradiography; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel; D010944:Plants / Q000382:microbiology; D012313:RNA / Q000235:genetics*; D000079962:RNA, Circular; D019244:RNA, Satellite; D012367:RNA, Viral / Q000235:genetics*; D014772:Viroids / Q000235:genetics*
chemical_list: D000079962:RNA, Circular; D019244:RNA, Satellite; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes