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Application of New DNA Sequencing Technologies for the Study of Epigenetic Abnormalities in Breast Cancer. John R. Edwards jre13@columbia.edu Columbia Genome Center July 25th, 2007. Sequencing by Synthesis. A new paradigm in genomic sequencing. DNA Polymerase Reaction.
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Application of New DNA Sequencing Technologies for the Study of EpigeneticAbnormalities in Breast Cancer John R. Edwards jre13@columbia.edu Columbia Genome Center July 25th, 2007
Sequencing by Synthesis A new paradigm in genomic sequencing
G - Linker - O-Block-3’ ACGCTAGCGATCATGCAGCTGCATCG TGCGATCG Sequencing by Synthesis Primer C T A Template
Structure of the Polymerase-DNA-Nucleotide Complex Pelletier et al. (1994) Science264, 1891-1903
DNA Sequencing by Synthesis (SBS) on a Chip Ju J, Li Z, Edwards JR, Itagaki Y. U.S. Patent (2003) 6,664,079
Molecular Structures of 3’-O-Allyl-dNTP-Allyl-Dye Nucleotide Analogues J. Ju et al. PNAS,2006, 103:19635-40.
CpG Methylation and Transcription Jones and Takai (2001) Science 293:1068-70
Fractionation Methods Show an Unbiased Coverage of the Genome McrBC = 3073 sequences RE = 2565 sequences
Categorical Breakdown of Methylated and Unmethylated Compartments
Methylation Limits the Effective Size of the Genome Small Genome Large Genome
DNA Methylation in Cancer Genomic Hypomethylation • Genomic instability? • Activation of proto-oncogenes? • Loss of Imprinting? M. Esteller (2005) Annual Review of Pharmacology and Toxicology 45:629-656.
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
Acknowledgements Jingyue Ju Timothy Bestor (Genetics and Development) Nicholas Turro (Chemistry) Victoria Haghighi (Psychiatry) Ju Lab James J. Russo Zengmin Li Lanrong Bi Xiaoxu Li Shundi ShiDae H. Kim Qingleng Meng Xiaopeng Bai Bestor Lab Rob Rollins Anne O’Donnell National Human Genome Research Institute/NIH NSF, Packard Foundation