IDENTIFYING GENOME CHANGES IN CANCER DEVELOPMENT

描述：（申请人摘要）人类疾病是由一种复杂的 DNA多态性或突变与环境因素的相互作用。 单核苷酸多态性（SNP）最近已被鉴定为 潜在的强大的基因分型手段，并预计 取代微卫星重复分析作为遗传学的标准 关联、连锁和作图研究。 因此，迫切需要 开发快速分析SNPs的强大工具。 本实验室 使用热稳定连接酶成功地检测和定量了广泛的 已知癌基因和肿瘤抑制基因的多态性和突变。 连接酶检测反应（LDR）结合多重PCR反应， 也已被用于检测人类的多种多态性， 识别. 因此，我们实验室开发的方法是理想的， 适用于快速评分的单核苷酸多态性在整个 基因组 该提案的第一个目标是扩展基于定量连接酶的 检测以在新型通用DNA阵列上对SNP进行评分。 为模特 系统，我们计划量化基因扩增或杂合性丢失， 结肠显微组织中c-myc、K-ras、APC和p53基因的表达 使用LDR和DNA阵列。 这种方法将用于帮助 在7 q31基因座上发现一个新的肿瘤抑制基因， 80%的大肠癌。 该提议的第二个目标是使用热稳定的内切核酸酶V 错配切割酶/热稳定DNA连接酶组合， 识别和精确表征未知编码区SNP。 这种snp 存在于已知的癌症基因中的频率很低，携带者处于 患乳腺癌、卵巢癌、前列腺癌或结肠癌的风险显着更高 癌 使用酶法鉴定单个或合并样本中的SNP 样本，我们希望大大加快发现SNPs在癌症 易感基因 酶发现/鉴定与基于通用阵列的 评分代表了一种综合方法， 核苷酸多态性的遗传研究，并可能最终导致一个 通过早期筛查和预防计划减少癌症负担。

DESCRIPTION: (Applicant's abstract) Human diseases arise from a complex interaction of DNA polymorphisms or mutations and environmental factors. Single nucleotide polymorphisms (SNPs) have recently been identified as potentially powerful means for genetic typing, and are predicted to supersede microsatellite repeat analysis as the standard for genetic association, linkage, and mapping studies. Thus, there is an urgent need to develop robust tools for the rapid analysis of SNPs. Our laboratory has used thermostable ligase to successfully detect and quantify a wide range of polymorphisms and mutations in known oncogenes and tumor suppressor genes. The ligase detection reaction (LDR) combined with multiplex PCR reactions, has also been used to detect multiple polymorphisms for human identification. Thus, the methods developed in our laboratory are ideally suited for rapid scoring of single nucleotide polymorphisms throughout the genome. The first goal of this proposal is to extend quantitative ligase-based detection to score SNPs on a new type of universal DNA array. As a model system, we plan to quantify gene amplification or loss of heterozygosity in the c-myc, K-ras, APC and p53 genes in microdissected tissue from colon tumors using LDR and DNA arrays. This approach will be used to help identify a new tumor suppressor gene at the 7q31 loci which is deleted in 80% of colorectal cancers. A second goal of this proposal is to use a thermostable Endonuclease V mismatch cleavage enzyme / thermostable DNA ligase combination to rapidly identify and precisely characterize unknown coding region SNPs. Such SNPs are present in known cancer genes at low frequency, and carriers are at a significantly higher risk of developing breast, ovarian, prostate or colon cancer. Using enzymatic methods to identify SNPs in single or pooled samples, we expect to greatly accelerate discovery of SNPs in cancer susceptibility genes. Enzymatic discovery/identification combined with universal array-based scoring represents an integrated approach to harness the power of single nucleotide polymorphisms for genetic studies, and may ultimately lead to a reduction of cancer burden through early screening and prevention programs.