Comparative Genomic HybridizationCGH HUJI比较基因组杂交CGH胡.ppt

Comparative Genomic Hybridization(CGH) Outline Introduction to gene copy numbers and CGH technology DNA copy number alterations in breast cancer (Pollack et.al., PNAS (2002)) Copy number polymorphism in human genomes (Sebat et.al., Science (2004)) Alteration in DNA Copy Number: amplification and deletion Alteration in DNA Copy Number: possible mechanism Array Based Comparative Genomic Hybridization Goal: to detect copy number alterations using a gene chip Ideally, the signal intensity is proportional to copy number Several genomes can be compared simultaneously Technical consideration in array CGH Hybridization signals Affected by base composition, repetitive sequences, chosen probes, saturation of the array, double-strand association etc. Lower signals obtained for lower complexity probes (cDNA and PCR products) Genome characteristics Hybridization of repetitive elements, should be blocked Copy number measurements Difficult to detect deletions Low-copy reiterated sequences Copy-number polymorphism Heterogeneous specimens (cells with altered DNA mixed with normal cells) Technical consideration in array CGH Specimen preparation Differences in quality of cell lines, frozen/fresh/fixed tissue Heterogeneous specimens Extraction of DNA Data analysis Significance of signal ratios Applications of array CGH in oncology Use in clinical trials for CLL drugs (to determine relationship between therapeutic options and genomic aberrations) Association of DNA copy-number with prognosis in a variety of tumors (prostate, breast, gastric, lymphoma) Detecting a region and not a gene Not always found in correlation with gene expression Wide range of genomic phenotypes Ongoing genomic instability results in heterogeneity Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors(Pollack et. al. (2002) PNAS 99:12963-8) Analysis of DNA copy number in breast tumors Array based CGH High resolution (gene-