Detection of G-Quadruplex DNA Using Primer Extension as a Tool

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.     

Development of a genotype‐by‐sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High‐throughput sequencing (HTS) technologies, such as genotype‐by‐sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization‐based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to “capture” specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300‐kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on‐target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer‐read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune‐related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.     

Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer.