Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/11/2004
Publication Date: 3/7/2005
Citation: Tzanetakis, I.E., Keller, K.E., Martin, R.R. 2005. The use of reverse transciptase for efficient first and second strand cdna synthesis from dsrna templates. Journal of Virological Methods. 124:73-77. Interpretive Summary: For many viruses, the only readily obtainable virus component is the double-stranded RNA that is produced during virus replication. Identification and characterization of these viruses is limited by the inability to obtain cDNA clones from double-stranded RNA. This paper describes an efficient method for cloning from dsRNA templates. The method has been applied to eight viruses from a range of hosts, including strawberry, blackberry, peas, and mint. Complete sequences were obtained for three viruses, and partial sequences were obtained for the other five viruses. The key to the method is the use of reverse transcriptase to make first- and second-strand DNA. In all other protocols in which people have tried to clone from dsRNA templates, reverse transcriptase is used to make first-strand DNA then DNA polymerase is used to make second-strand DNA. It is thought that the reverse transcriptase is not affected by plant inhibitors that are copurified with the dsRNA whereas DNA polymerase is inhibited.
Technical Abstract: Molecular characterization of eight distinct, difficult-to-clone, RNA plant viruses was accomplished after the development of a reverse transcriptase-based first- and second-strand cDNA synthesis method. Double-stranded (ds) RNA templates isolated from strawberry and blackberry, as well as from several herbaceous hosts (mint, pea and tobacco) were cloned using this method. Templates were combined with random primers then denatured in methyl mercuric hydroxide. Reverse transcriptase was added and the reaction was then incubated at 50ºC, followed by addition of RNase H and further incubation at 37ºC. The resulting dsDNA was then digested with restriction endonucleases to produce shorter fragments that could be cloned efficiently with a T-tailed vector after adding an A overhang using Taq polymerase. This procedure resulted in a high number of cloned fragments and allowed insert sizes up to three kilobase-pairs. Unlike traditional cDNA construction methods, there is no need for additional enzymes/steps for second-strand synthesis, PCR amplification or prior sequence information. Synthesis and cloning of cDNA derived from dsRNA templates is much more efficient than with previously described methods. This procedure also worked well for cloning gel purified dsRNA and with single-stranded RNA templates.