Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

DNA-dependent RNA polymerase realignment is sensitive to the relative concentration of the substrate specified by the slippage site and by the DNA template base 5 adjacent to it (47) . To assess whether this also pertains with Reverse Transcriptase realignment, different dNTP concentration ratios were assayed. Nine dNTP ratio combinations with 5, 50 or 500 M for the dTTP (specified by the A-tract slippage motif) and 5, 50 or 500 M for the dGTP (specified 5 adjacent to the template motif) were tested; the dATP and dCTP substrates were each present at 500 M. Presence of the 'model' WT stem-loop stimulated addition of T ( Figure 1D , lanes 4-9); this stimulation was increased with higher At the highest dTTP concentration tested, a modest level of base addition is also observed but only at the highest ratio of [dTTP]:[dGTP] (lane 16). These results indicate that the RNA stem-loop is a strong stimulator for RT SuperScriptâ„¢ III realignment directionality, promoting addition of an extra T in the cDNA, but not the omission of a T. Interestingly, in absence of the RNA stem-loop structure, realignment directionality is the inverse. This directionality difference indicates that the RNA stem-loop is also a strong inhibitor for omission of a base complementary to a template base. In summary, realignment efficiency and directionality is influenced by the relative dNTP concentrations and by RNA template structure. products derived from the cDNA template, and of the PCR products derived from the synthetic DNA template. Standard LPE products (whose synthesis did not involve slippage and reflect the original length of the motif in the chemically synthesized template) are indicated by an orange arrowhead.