CORE--MOLECULAR ANALYSIS AND HIGH THROUGHPUT GENOTYPING

描述（由申请人提供）：分子流行病学和高 Genotyping Core（简称为Molecular Core）将提供 为项目1（卡罗莱纳乳腺癌研究：DNA修复）提供实验室支持 基因和乳腺癌风险），项目2（卡罗莱纳乳腺癌研究： 乳腺癌发展中的雌激素受体改变）和项目3 (Long岛屿乳腺癌研究：氧化应激和乳腺癌风险） 在人口科学计划，和项目5（ 对新辅助化疗有反应的分子标志物） SPORE的转化科学计划。 分子核心进行广泛的基于PCR的体细胞遗传分析 从福尔马林固定的肿瘤切片中获得的DNA和基于PCR的基因分型 从外周血中纯化的生殖系DNA。在其八年的运作中， 核心已经设计并实施了鉴定基因突变的试验， 缺失和扩增，杂合性丢失，微卫星 不稳定性，重复DNA区域中复杂的种系变异，如 H-ras可变数目串联重复序列与基因的生殖系多态性 参与细胞过程，如DNA修复，致癌物质和激素 代谢和氧化代谢。 该设施的集中性质简化了实验功能 因为可以对不同的项目执行相同或类似的分析。 集中化确保了经验丰富的人员，最高效率， 严格的质量控制，实验室资源的经济利用， 通过实验室人员的交叉培训实现技术的多功能性，以及 集中数据库和组织跟踪程序。而且 核心解决了大规模临床和 通过设计分析算法， 简化分子任务，提高内部和之间的再现性 数据集，开发自动化方法以提高样品通量 而不损失灵敏度或特异性，并优化技术， 允许分析微量或次优质量的DNA 从石蜡包埋组织中获得的模板。 为了支持拟议的项目，分子核心将评估一个 新描述的雌激素受体α突变，并将应用新的 提高样品通量的技术进步，包括基于Taqman的 DNA修复酶、氧化代谢中基因分型变异的测定 酶和CYP 19/芳香化酶，以及Affytoplasmp 53基因芯片 用于检测p53点突变的寡核苷酸阵列筛选方法。 分子核心还将协助组织采购和分析核心 (Core 3）在免疫组织化学染色测定的发展中， 基于基因表达谱鉴定合适的组织对照。 最后，该核心将成为UNC-CH基因发现工作的资源， 其合作者在新发现的DNA修复基因的应用中， 多态性与SPORE的群体研究有关。

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.