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RNArticles
World's Longest Cloned RNA... Almazan, F., Gonzalez, J.M., Penzes, Z., Izeta, A., Calvo, E., Plana-Duran, J., and Enjuanes, L. 2000 "Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome." PNAS 97, 5516-5521. Lai, M.M.C. (2000) "The making of infectious viral RNA: no size limit in sight." PNAS 97, 5025-5027. Recombinant nucleic acid technology has progressed substantially over the last 20 years. Historically, cloning small nucleic acid molecules of 5 kb or less challenged researchers. Persevering scientists have overcome major limitations to yield the recent results reported by Almazan et al.: the successful cloning of the 27 kb viral genome of TGEV, a porcine coronavirus. Several obstacles stand in the way of cloning large RNA molecules. The first difficulty is making a faithful cDNA copy of the RNA. The advent of high fidelity reverse transcriptases and DNA polymerases has enabled the accurate synthesis of cDNA molecules from RNA templates. Secondly, "poison sequences" in the RNA lead to intrinsically weak plasmids containing the cDNA copy. Almazan et al. avoided this problem by cloning the viral genome in pieces and subsequently ligating the segments together. The deleterious sequences were added in the final cloning step, thereby eliminating the need to grow unstable intermittent plasmids. Finally, traditional cloning vectors have proven too small to accommodate such large nucleic acids. A BAC (bacterial artificial chromosome) solved this quandary. Sequences up to 150 kb have been successfully inserted into BACs, which is readily grown in culture. These advancements in recombinant nucleic acid technology allow for further investigation of large viral RNAs with the promise of elucidating their function. |