Abstract
DNA sequence and structure play a key role in imparting fragility to different regions of the genome. Recent studies have shown that non-B DNA structures play a key role in causing genomic instability, apart from their physiological roles at telomeres and promoters. Structures such as G-quadruplexes, cruciforms, and triplexes have been implicated in making DNA susceptible to breakage, resulting in genomic rearrangements. Hence, techniques that aid in the easy identification of such non-B DNA motifs will prove to be very useful in determining factors responsible for genomic instability. In this study, we provide evidence for the use of primer extension as a sensitive and specific tool to detect such altered DNA structures. We have used the G-quadruplex motif, recently characterized at the BCL2 major breakpoint region as a proof of principle to demonstrate the advantages of the technique. Our results show that pause sites corresponding to the non-B DNA are specific, since they are absent when the G-quadruplex motif is mutated and their positions change in tandem with that of the primers. The efficiency of primer extension pause sites varied according to the concentration of monovalant cations tested, which support G-quadruplex formation. Overall, our results demonstrate that primer extension is a strong in vitro tool to detect non-B DNA structures such as G-quadruplex on a plasmid DNA, which can be further adapted to identify non-B DNA structures, even at the genomic level.
Highlights
DNA exists majorly in the B form except in certain regions of the genome such as telomeres, wherein G-quadruplex structures have been identified [1,2]
Further we show that pause sites corresponding to the G4 motif depend on the concentration of DNA template, presence of cations, which are known to support G-quadruplex formation and the position of the primers used
Formation of non-B DNA structures like G-quadruplex, have been suggested to arrest physiological processes such as replication and transcription [18,19]. We tested whether such polymerase arrest due to formation of altered DNA structures can be detected using primer extension, when the region of interest is present on a double-stranded plasmid DNA (Fig. 1A)
Summary
DNA exists majorly in the B form except in certain regions of the genome such as telomeres, wherein G-quadruplex structures have been identified [1,2]. The presence of pause sites due to the formation of G-quadruplex structure at the BCL2 mbr was studied on pMN7 by primer extension using the oligomer SCR105. We tested whether such polymerase arrest due to formation of altered DNA structures can be detected using primer extension, when the region of interest is present on a double-stranded plasmid DNA (Fig. 1A).
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