Application of New DNA Sequencing Technologies for the Study of Epigenetic Abnormalities in Breast Cancer John R. Edwards Columbia Genome.

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Presentation transcript:

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