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.

Standard synthesis of the cDNA transcript is achieved by successive nt addition at the 3 end of the cDNA transcript. The RT is in its pre-translocated state when incorporation of the substrate occurs to yield the 3 end of the cDNA in the polymerase's catalytic center ( Figure 6A , C and E). After RT translocates one nt forward to the next template base, and its catalytic center is free of the cDNA 3 end, the RT is in its post-translocated state ( Figure 6B and (51) . This RNA was chemically synthesized. (B) LPE analysis of the RT-PCR (using cDNA as template) and PCR (using synthetic DNA as template) products, were performed with similar strategy as shown in Figure 1 using specific primers. 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. D). In absence of substrate in the catalytic centre, RT can oscillate between post-translocation and pre-translocation conformations ( Figure 6 , sets A and B, C and D).