Application of New DNA Sequencing Technologies for the Study of Epigenetic Abnormalities in Breast Cancer John R. Edwards Columbia Genome Center July 25th, 2007
Sequencing by Synthesis A new paradigm in genomic sequencing
DNA Polymerase Reaction
DNA Polymerase Reaction Modifications
G - Linker - O-Block-3’ Sequencing by Synthesis ACGCTAGCGATCATGCAGCTGCATCGACGCTAGCGATCATGCAGCTGCATCG TGCGATCGTGCGATCG Template Primer C A T
Structure of the Polymerase-DNA-Nucleotide Complex Pelletier et al. (1994) Science 264,
Ju J, Li Z, Edwards JR, Itagaki Y. U.S. Patent (2003) 6,664,079 DNA Sequencing by Synthesis (SBS) on a Chip
J. Ju et al. PNAS, 2006, 103: Molecular Structures of 3’-O-Allyl-dNTP- Allyl-Dye Nucleotide Analogues
Emulsion PCR Based DNA Template Preparation
Methylation Landscape of the Human Genome
CpG Methylation and Transcription Jones and Takai (2001) Science 293:
CpG Composition of the Human Genome
Fractionation of the Genome
McrBC and RE Digests
Fractionation and Analysis Pipeline
Fractionation Methods Show an Unbiased Coverage of the Genome McrBC = 3073 sequences RE = 2565 sequences
Custom Methylation Tracks on the UCSC Genome Browser
Methylation Status of Promoters
Alu Elements are Highly Methylated
Categorical Breakdown of Methylated and Unmethylated Compartments
Methylation Limits the Effective Size of the Genome Small Genome Large Genome
Whole-genome Profiling of Breast Cancer
DNA Methylation in Cancer M. Esteller (2005) Annual Review of Pharmacology and Toxicology 45: Genomic Hypomethylation Genomic instability? Activation of proto-oncogenes? Loss of Imprinting?
New Tools to Probe DNA Methylation in Breast Cancer Technology Requirements Whole genome approach Must examine state of repetitive elements Unbiased Must be useable for primary tumors Goals Characterize complete methylation profile of breast cancer Compare normal/tumor, tumor/tumor, tumor/cell-line Investigate potential as biomarker Understand patterns of global hypomethylation and regional hypermethylation
Whole-Genome Methylation Profiling
Acknowledgements Acknowledgements Jingyue Ju Timothy Bestor (Genetics and Development) Nicholas Turro (Chemistry) Victoria Haghighi (Psychiatry) Ju Lab James J. Russo Zengmin Li Lanrong BiXiaoxu Li Shundi ShiDae H. Kim Qingleng MengXiaopeng Bai Bestor Lab Rob Rollins Anne O’Donnell National Human Genome Research Institute/NIH NSF, Packard Foundation