IDENTIFYING GENOME CHANGES IN CANCER DEVELOPMENT

描述：(申请人摘要)人类疾病源于一种复合体 DNA多态或突变与环境因素的相互作用。 单核苷酸多态(SNPs)最近被发现是 潜在的强大的基因分型手段，预计 取代微卫星重复序列分析作为遗传分析的标准 关联性、连锁性和图谱研究。因此，迫切需要 开发强大的工具来快速分析SNPs。我们的实验室有 使用耐热连接酶成功地检测和定量了广泛的 已知癌基因和抑癌基因的多态和突变。 连接酶检测反应(LDR)与多重PCR反应相结合， 也被用来检测人类的多个多态 身份证明。因此，我们实验室开发的方法是理想的 适用于单核苷酸多态的快速评分 基因组。 这项提议的第一个目标是扩展基于量化连接酶的 检测一种新型通用DNA阵列上的SNPs。作为一名模特 系统中，我们计划量化基因扩增或杂合性丢失 C-myc、K-ras、APC和P53基因在结肠显微解剖组织中的表达 使用LDR和DNA阵列的肿瘤。这种方法将被用来帮助 在7q31位点发现一个新的肿瘤抑制基因，该基因在 80%的结直肠癌。 这项提议的第二个目标是使用耐热核酸内切酶V 错配裂解酶/耐热DNA连接酶组合快速 识别和准确鉴定未知编码区的SNPs。这样的SNPs 在已知的癌症基因中以低频率存在，携带者处于 患乳腺癌、卵巢癌、前列腺癌或结肠癌的风险显著升高 癌症。用酶标法鉴定单个或混合的SNP 样本，我们预计将大大加快癌症中SNPs的发现 易感基因。 酶发现/鉴定与通用阵列相结合 得分代表了一种利用单打力量的综合方法 用于遗传学研究的核苷酸多态，并可能最终导致 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.