Dissertation/Thesis Abstract

Sequencing-based high-throughput profiling of DNA methylation
by Ball, Madeleine Price, Ph.D., Harvard University, 2010, 275; 3415411
Abstract (Summary)

DNA methylation is an epigenetic mark found in mammals that is implicated in inhibiting gene transcription, modulating genome behavior within cells in a mitotically heritable manner. To study this phenomenon in a high-throughput manner, two different and complementary methods are presented here: Bisulfite Padlock Probes (BSPPs) and Methyl Sensitive Cut Counting (MSCC). BSPPs are microarray-synthesized oligonucleotides that isolate -10,000 genomic locations in bisulfite-treated DNA in a highly specific manner, allowing highly accurate profiling of a targeted subset of genomic locations. MSCC is an untargeted method that uses a methylation sensitive restriction enzyme, HpaII, to quantitatively profile methylation at all uniquely identifiable CCGG DNA sequences (-1.4 million CpG sites in human).

Both methods revealed a pattern of higher methylation in the gene bodies of highly expressed genes in lymphocyte cell lines (MSCC and BSPP) and fibroblasts (BSPP only), building upon growing evidence for a phenomenon of gene body methylation. MSCC was then applied to profile in vivo methylation changes in mouse neural tissue; this highly demanding task accurately detected moderate levels of methylation changes in a small set of locations (2-3% of sites with >=20% change), demonstrating MSCC's ability to provide accurate, genome-scale methylation measurements. The same experiments failed to detect gene body methylation in this tissue, indicating that the phenomenon is not universal and may be limited to contexts. Both BSPP and MSCC use high-throughput sequencing, providing tools for methylation profiling that take advantage of the rapidly dropping costs of DNA sequencing to profile DNA methylation in an accurate, quantitative manner.

Indexing (document details)
Advisor: Hogle, James
Commitee:
School: Harvard University
School Location: United States -- Massachusetts
Source: DAI-B 71/07, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Genetics, Bioinformatics
Keywords: DNA methylation, Epigenetics, Genome analysis, Next-generation sequencing
Publication Number: 3415411
ISBN: 9781124090917
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