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

Abstract

In this communication, we present data which describe optimum conditions for reverse transcription of large ribonucleic acid (RNA) templates into deoxyribonucleic acid (DNA) transcripts by the avian retrovirus reverse transcriptase in vitro. In contrast to previous studies, we have optimized all of the reaction components 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 herein for the uninterrupted reverse transcription of large RNA templates (greater than 1000--2000 nucleotides) are superior to those described previously because they are reproducible, do not require the presence of ribonuclease inhibitors, and do not result in the precipitation of components of the reaction mixture during incubation.