Enzymic synthesis of deoxyribonucleic acid by the avian retrovirus reverse transcriptase in vitro: optimum conditions required for transcription of large ribonucleic acid templates

Abstract

Optimum conditions for reverse transcription of large RNA templates into DNA transcripts by the avian retrovirus reverse transcriptase in vitro are described. In contrast to previous studies, all of the reaction components were optimized with respect to their influence on the size of DNA transcripts rather than the incorporation of radio-labeled deoxynucleoside triphosphates into acid-insoluble DNA product. The most dramatic effect on uninterrupted reverse transcription is the presence of physiological concentrations (i.e., 148 mM) of monovalent cation in the reaction mixture, although all of the components of the reaction influence the size of the DNA transcripts synthesized to some extent. The enzymatic conditions described for the uninterrupted reverse transcription of large RNA templates (> 1000-2000 nucleotides) are superior to those described previously because they are reproducible, do not require the presence of RNase inhibitors, and do not result in the precipitation components of the reaction mixture during incubation.