Bisulfite Fragmentation at Non-B Motifs in Genomic DNA
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The frequency of non-B DNA motifs in a genome steadily increases as the epigenetic potential of the organism increases. This demonstrates that non-B structures are under positive selective pressure and are not only related to harmful effects, but may also play an epigenetic role in developmentally complex organisms. This summer we investigated these non-B structures for both their association with harmful genetic mutation and for their regulatory potential in genomic DNA. Previously published work notes that a non-B secondary structure in the DNA of breast-cancer cells rendered it bisulfite accessible under non-denaturing conditions suggesting that bisulfite can break native DNA preferentially at sites of secondary structure. We developed a cloning method with the goal of isolating sequences flanked by closely spaced secondary structures in human prostate cancer cell lines. By treating the DNA with bisulfite under non-denaturing conditions, cloning and sequencing these isolated DNA fragments we expected to find non-B recurring motifs flanking these sequences. About half of the sequences recovered were found to originate from G+C rich regions of the prostate cancer genome, and several sequences were found to be flanked by the expected motifs including one sequence that was recovered three separate times on the NT_026437.12 contig of chromosome 14 which is now under further study. Our results suggest that these formations can be isolated from cancer cells. Future work will determine whether or not these sequences can be utilized as biomarkers in a tumor-specific fingerprint.