Application of New DNA Sequencing Technologies for the Study of

EpigeneticAbnormalities in Breast Cancer

John R. Edwardsjre13@columbia.edu

Columbia Genome CenterJuly 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 SynthesisACGCTAGCGATCATGCAGCTGCATCG

TGCGATCG

Template

Primer

C

AT

Structure of the Polymerase-DNA-Nucleotide Complex

Pelletier et al. (1994) Science 264, 1891-1903

Ju J, Li Z, Edwards JR, Itagaki Y. U.S. Patent (2003) 6,664,079Ju 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, 103:19635-40.

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 293:1068-70

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

http://epigenomics.cu-genome.org/html/meth_landscape/

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 45:629-656.

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

AcknowledgementsAcknowledgements

Jingyue Ju

Timothy Bestor (Genetics and Development)Nicholas Turro (Chemistry)Victoria Haghighi (Psychiatry)

Ju LabJames J. Russo Zengmin Li Lanrong Bi Xiaoxu Li Shundi Shi Dae H. Kim

Qingleng Meng Xiaopeng Bai

Bestor Lab Rob RollinsAnne O’Donnell

National Human Genome Research Institute/NIHNSF, Packard Foundation