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.

The LPE results showed that by increasing the distance between the model stem-loop and the A7 tract by just one nt, the stimulatory effect of the RNA stem-loop structure on base addition is abolished. The results also show that the inhibitory effect of the stem-loop on base omission is abolished as well. Base omission is now more sensitive to dNTP concentration ratio variation. This is most evident with a higher concentration of the dGTP substrate (specified by the template base 5 adjacent to the A-tract motif), than that of the dTTP substrate (specified by the slippage motif) (Supplementary Figure S2A and C). The results with E. coli RNA polymerase generated RNA, showed that the model stem-loop 5 adjacent to an A5 motif does not, at equimolar dNTP, stimulate SuperScriptâ„¢ III-mediated base addition (data not shown). Interestingly, similar experiments using derivative constructs specifying the model stem-loop 0, 1, 2, or 3 nt 5 to A5 motif, showed that though the RNA stemloop does not stimulate base addition on A5, its inhibitory effect on base omission is present when the model stemloop is 5 adjacent to the A5 (Supplementary Figure S2B and C). The distance between the 'road-blocking' structure formation and the A7 slippage motif was also explored by antisense RNA experiments (Supplementary Results and Supplementary Figure S3) .