Patterns of modified cytosines (5-methylcytosine and 5-hydroxymethylcytosine) are required for the normal function of the genome, and perturbations of patterns of modified cytosine can be lethal and lead to gross changes in patterns of gene expression. Methylation patterns are grossly abnormal in many forms of human cancer. Existing methods of genome-wide methylation profiling require large amounts of DNA, overestimate methylation levels, and cannot cover the entire genome. We are developing a method wherein enzymes are used to modify sites of methylation and hydroxymethylation with chemical tags that render these sites easily detectible by nanopore sequencing by synthesis and other platforms for single molecule DNA sequencing. The result will be a comprehensive method for the detection of modified bases in human genomes with much greater sensitivity, accuracy, economy, and throughput than existing methods.
The function of the genome and the regulated expression of many genes depends on patterns of methylated and hydroxymethylated cytosines across the entire genome, and especially in the promoter regions of genes. These patterns are abnormal in a number of human disorders, with cancer genomes showing the most severe derangements. It has not been previously possible to obtain whole genome patterns of modified cytosines at single nucleotide resolution with acceptable levels of accuracy, sensitivity, and economy; we propose to develop a radically new method that will map all modified cytosines with much greater efficiency than any existing method. We will then be able to determine the contribution of epigenetic effects to any human disorder.
| Li, Xiaoxu; Erturk, Ece; Chen, Xin et al. (2018) Photochemical conversion of a cytidine derivative to a thymidine analog via [2+2]-cycloaddition. Photochem Photobiol Sci 17:1049-1055 |
