Peliska JA, Benkovic SJ.

Biochemistry. 1994 Apr 5;33(13):3890-5 doi:10.1021/bi00179a014

Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

When the extra base added by the RTase in a template-switching reaction is determined by sequencing, the result does not reflect previous results obtained by primer extension assay (where A >> C on non-capped blunt DNA/RNA ends). This may be because of the differential efficiency of the RTase to extend a template over various single-base mismatches. In this assay, T to C and G to C were more frequent than T to A and G to A. Nevertheless, the experiments support previous evidence that addition is mostly limited to a single nucleotide. RT reaction: 50 mM Tris-HCl, pH 8.0; 75 mM KCl;, 0.1 mM EDTA; 1 mM DTT; 0.1% Triton X-100; 100 µM each dNTP; 7 mM MgCl2; 200 nM 24-base DNA-40-base RNA primer-template; 700 nM 41-base RNA template 2; 700 nM 42-base RNA template 3; and 100 nM HIV-1 RT in a final volume of 10 µL. When reaction products were to be sequenced, mixtures were incubated at 37 °C for 2 h.