BRIDGING THE GAP: PROBING THE EFFECTS OF DNA METHYLATION ON BINDING DYNAMICS THROUGH NOVEL METHODOLOGY, AND HISTORICAL REVIEW
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Date
2015-12-11
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Johns Hopkins University
Abstract
Though more than a decade has passed since the completion of The Human Genome Project, the knowledge derived from it has had little impact on our understanding of human health. The reason for this knowledge gap is that only 6.5-10% of the human genome appears to have direct functionality, with the remainder of functional genome elements existing hidden within “junk DNA”. Much effort has been directed towards identifying such regulatory components, like the ENCyclopedia of DNA Elements (ENCODE) project, however, such studies are incapable of distinguishing specific from non-specific protein-DNA binding, or intrinsic vs. environmentally affected binding affinity.
A new method of producing randomly methylated DNA libraries is presented here which should help bridge this knowledge gap. Using mobility shift assay (EMSA), cleavage-qPCR, and immunoprecipitation sequencing (IP-Seq), this method was used to probe the activity and binding strength between Type II restriction endonucleases and randomly methylated DNA. The generation of randomly methylated libraries of genetic material will enable future studies of protein binding affinity, relative to both genetic sequence, and the presence of local and distant DNA methylation. Such experiments can integrate further elements of epigenetic complexity into current methods for identifying binding motifs of proteins, and provide further insight into the underlying mechanisms of epigenetic modification.
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Epigenetics, Review