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.

When the leading edge of the RT polymerase encounters relevant template RNA structure ( Figure 6 , H), its progression is restrained at the pre-translocation state ( Figure 6C ) increasing the propensity for backward realignment of the cDNA 3 end. When the cDNA: RNA hybrid contains an appropriately positioned slippage-prone sequence there is pairing potential in the backward realigned cDNA (at least its 3 end). Such a 1 nt backward realignment would mimic the situation where the RT polymerase is in the posttranslocation state ( Figure 6G ). The template base in the RT catalytic centre is available for pairing with the substrate, and so productive substrate incorporation that yields slippage-mediated base addition ( Figure 6 , from G to C after incorporation of the substrate).