Cycling of RNA-directed DNA polymerase on natural and synthetic RNA templates

RNA-DIRECTED DNA polymerase (reverse transcriptase) catalyses in vivo the synthesis of DNA complementary to the RNA of tumour viruses. The DNA is integrated into the host genome and is therefore implicated in the subsequent tumorigenesis 1 . The mechanism by which reverse transcriptase replicates the viral RNA into complementary DNA (cDNA) and its resultant integration, as well as the interaction of the viral components in the host, remain unclear 2 . Reverse transcriptase has been used in vitro to synthesise cDNA to mRNA; this cDNA can be used to identify and characterise the cellular processes dependent on nucleic acids. The conditions of the reactions have been optimised 3,4 ; however problems remain with both the quality and quantity of the cDNA 5,6,15 . We report here in vitro conditions such that each RNA-directed DNA polymerase molecule synthesises several to more than 100 transcripts, depending on whether natural or synthetic templates are used. As noted previously 3,4 , the fidelity of the cDNA transcript, the amount, and the time course of polymerisation depend strongly on the reaction conditions.