Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 16, 2004
Publication Date: June 11, 2004
Citation: Tzanetakis, I.E., Keller, K.E., Martin, R.R. A simplified cdna cloning method for recalcitrant double-stranded rna viral templates using reverse transcriptase. Phytopathology. 2004. v94 p. S104. Technical Abstract: An unknown virus of pea, which had proven unclonable by conventional methods, was characterized after the development of a novel cloning method. Using dsRNA viral templates and random primers, reverse transcriptase was used to generate first-strand cDNA. Second-strand synthesis was performed using a combination of reverse transcriptase and RNaseH with restriction enzyme(s) added to give shorter fragments that are more readily cloned. Taq DNA polymerase was used to A-tail the cDNA which was then cloned into a T-tailed cloning vector. Using this method, sequence data has been obtained from five separate viruses isolated from three different host plants. This cloning method resulted in a high number of cloned fragments, with clone sizes up to 1.5 kbp. Unlike traditional cDNA cloning methods, this procedure does not use DNA polymerase I and DNA ligase, making it much more efficient and economical. Finally and most important, it allows cloning of cDNA derived from dsRNA templates, whose cloning by conventional methods had not been successful.