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.

LPE analysis was performed with primer, R 821, complementary to the sequence immediately adjacent to the A-tract in one DNA strand of the (RT)-PCR product. An acyC terminator mediates LPE termination at the site specified by the template base position underlined in the sequence 5 -G-spacer-A's-3 ( Figure 3B, left) . The varied spacer lengths (0,1,2,3) determine the staggered LPE product sizes, markers, seen on the gel. The shift is related to the 1 nt length difference of the spacers involved ( Figure 3B , PCR). With SuperScriptâ„¢ III RT, at equimolar dNTP the RT-PCR derived LPE products from all four constructs contain detectable base addition ( Figure 3B, lanes 1-4) . With dNTP ratio conditions that favor base addition, both the efficiency of addition and number of bases added, increase with spacer length extensions ( Figure 3B , lanes 5-8). In contrast, with dNTP ratio conditions that favor base omission, both the efficiency of base absence and number of bases missing, decreases with spacer length extensions (Figure 3B, lanes 9-12) .