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.

With the wtSL-A7 construct, reverse transcription using SuperScriptâ„¢ III enzyme and all dNTPs present at 500 M, showed strong realignment-mediated addition of an extra A ( Figure 1D , lane 9) but no addition with the MUTsl-A7 construct, where the potential for base-pair formation is greatly diminished ( Figure 1D , lane 18). The corresponding control LPE marker showed no slippage addition for both the WT ( Figure 1C and D, lane 19) and mutant con- The subsequent analysis schemes are below each cartoon. LPE analysis (orange) involved a labeled primer that anneals 3 adjacent to the slippage motif and terminates at the base 5 adjacent to the motif. (C) LPE marker controls for DNA polymerase slippage. Chemically synthesized DNA counterparts of the corresponding RNA were used to generate a PCR product that served as template for LPE analysis. (D) LPE analysis with acyG terminators. 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. structs ( Figure 1C and D, lane 20). These LPE markers indicate that the DNA polymerase reagents (i.e. Taq and Vent exo-polymerases) are not responsible for the base addition. However, the wtSL-A7 and MUTsl-A7 constructs do show some omission of an A base ( Figure 1D , lanes 9 and 18) with a similar signal detection level as the corresponding LPE markers ( Figure 1D, lanes 19 and 20) .