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.

Initial experiments investigating a possible role for stemloops in stimulating indel formation utilized SuperScript™ III, the widely used genetically engineered RT and two chemically synthetized 75 nt RNA constructs containing 7A's. These specified the WT, or a variant, of the 'model' RNA stem-loop structure 5 adjacent to 7A's. The first construct 'wtSL-A7' has the WT sequence specifying the 'model' stem-loop 5 -GCGGGCgcaaGCCCGC-3 , with the potential of base pairing indicated in upper case. The second 'MUTsl-A7' has the 5 side sequence of the stem substituted by complementary nt bases, i.e. from 5 -GCGGGC-3 to 5 -CGCCCG-3 to prevent potential formation of the model stem-loop structure ( Figure 1A and B). RT reactions were performed with all dNTP equimolar at 500 M. The cDNAs were then amplified by PCR with Taq polymerase to yield the 'RT-PCR products'. The controls for Taq polymerase slippage used two chemically synthetized 75 nt DNAs, whose sequence corresponds to that of the test RNA sequence, used as template for PCR amplification. This yields the 'PCR products' referred to below. Next, the two RT-PCR and the two PCR products were used as templates for Limited Primer Extension (LPE) analysis for detecting the addition or omission of a base(s) in the T/Atract derived sequence. LPE reactions were performed with one primer whose sequence is complementary to the template sequence adjacent to the T-tract present in one of the two strands of the RT-PCR and PCR products [the other DNA strand has the corresponding A-tract]. The conditions of the LPE reaction enable the primer to be extended to the first template base position at which termination was arranged to occur by incorporation of an acyclic dGTP (acyGTP) base. This leads to efficient termination at the first base C of the template encountered by the polymerase during extension of the primer as the corresponding dGTP standard substrate is absent from the reaction (see Materials and Methods). The C at which LPE termination occurs is 5 adjacent to the T-tract (other sites and acyclic dNTPs are used as controls in Supplementary Data). The length of the LPE product also depends on the occurrence of any indel in the T-tract motif. In absence of slippage of the DNA polymerases used for amplification of the chemically synthesized DNA (Taq polymerase control) and subsequently for generation of the LPE product (Vent exo − polymerase), a homogeneous length LPE product is expected. This is used as a length marker. Comparison of the pattern of the LPE product(s) generated from RT-PCR with the marker reveal specific RT polymerase slippage-mediated base indels (Figure 1C ).