DEVELOPMENT OF RESTRICTION ENZYME-BASED ASSAYS FOR CONCURRENT DETECTION OF MULTIPLE CYTOSINE MODIFICATIONS AND GENOMIC ABERRATIONS

Abstract

Epigenetic modifications that regulate gene expression are essential for normal growth and could be aberrated in disease state. Gold standard bisulfite-based methods to detect DNA methylation are harsh and cause severe DNA damage. Hence, we developed restriction enzyme-based DARE assay that combines MspI/HpaII isoschizomer to simultaneously detect methylation and genomic aberrations in both small quantity of DNA and single cells (scDARE). However, DARE similar to bisulfite-based methods, unable to differentiate functionally contrasting methylation and hydroxymethylation modifications. To this end, we developed DARESOME assay that detects 5C, 5mC and 5hmC in a single assay. We further adapted this assay to be applied in cell-free DNA, cfDARESOME. Beyond being the first quantitative 5hmC assay for cfDNA, cfDARESOME provide genomic information such as CNA and SNVs as well. Overall, this thesis presents multiple novel multimodal assays that can provide concurrent genomic and epigenomic information for several important applications.