CORE--MOLECULAR ANALYSIS AND HIGH THROUGHPUT GENOTYPING

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DESCRIPTION (provided by applicant): The Molecular Epidemiology and High Throughput Genotyping Core (referred to as the Molecular Core) will provide laboratory support for Project 1 (Carolina Breast Cancer Study: DNA Repair Genes and Breast Cancer Risk), Project 2 (Carolina Breast Cancer Study: Estrogen Receptor Alterations in Breast Cancer "Development) and Project 3 (Long Island Breast Cancer Study: Oxidative Stress and Breast Cancer Risk) within the Population Sciences Program, and Project 5 (Correlation of Molecular Markers with Response to Neoadjuvant Chemotherapy) within the Translational Sciences Program of the SPORE. The Molecular Core performs a wide range of PCR-based somatic genetic analyses on DNA obtained from formalin-fixed tumor sections and PCR-based genotyping on germline DNA purified from peripheral blood. In its eight years of operation, the Core has designed and implemented assays to identify gene mutations, deletions and amplification, loss of heterozygosity, microsatellite instability, complex germline variation in repetitive DNA regions such as the H-ras variable number tandem repeat, and germline polymorphisms in genes involved in cellular processes such as DNA repair, carcinogen and hormone metabolism, and oxidative metabolism. The centralized nature of this facility streamlines experimental functions since the same or similar analyses may be performed for different projects. Centralization ensures highly experienced personnel, peak efficiency, stringent quality control, economic utilization of laboratory resources, technical versatility through cross training of laboratory personnel, and centralization of database and tissue tracking procedures. Furthermore, the Core has addressed the particular challenges of large-scale clinical and population-based molecular studies by designing analytical algorithms that streamline the molecular tasks and enhance reproducibility within and between data sets, developing automated approaches to increase sample throughput without loss of sensitivity or specificity, and optimizing techniques to permit analysis of the minute quantities or sub-optimal quality of DNA template obtained from paraffin-embedded tissues. In support of the proposed projects, the Molecular Core will evaluate a newly-described mutation in estrogen receptor-alpha, and will apply new technologic advances that increase sample throughput, including Taqman-based assays for genotyping variants in DNA repair enzymes, oxidative metabolism enzymes and the CYP19/aromatase enzyme, and the Affymetrix p53 GeneChip oligonucleotide array screening approach for detecting p53 point mutations. The Molecular Core will also assist the Tissue Procurement and Analysis Core (Core 3) in the development of immunohistochemical staining assays by identifying appropriate tissue controls based on gene expression profiles. Finally, this Core will be a resource for gene discovery efforts by UNC-CH and its collaborators in the application of newly-identified DNA repair gene polymorphism to the population studies of the SPORE.