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 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.