Fate of direct and inverted repeats in the RNA hypermutagenesis reaction

Abstract

RNA hypermutagenesis results from cDNA synthesis in the presence of highly biased dNTP precursor concentrations and preferentially exploits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Such reaction conditions slow down DNA synthesis, which might be conducive to strand transfer and deletion. This has been investigated. A 6 bp inverted repeat nested between 10 bp repeats was efficiently deleted at dCTP concentrations typically used. Inter- or intramolecular strand transfer between 10 bp repeated sequences separated by runs of templated G residues occurred, but at lower concentrations. If RNA hypermutagenesis of a sequence containing direct and inverted repeats is unavoidable, avian myeloblastosis virus (AMV) reverse transcriptase could be used, as strand transfer occurs with much diminished dCTP substrate dependence.