FOUR COLOR ARRAY BOUND SNP/MUTATION DETECTION IN CANCER

描述：（申请人的描述） 开发具有成本效益的高通量采样， 平行的DNA分析方法将有助于正常人的分子分析。 和病态的国家此R21/R33计划侧重于检测单个 核苷酸多态性（SNP）和癌症中的点突变。我们的目标是 R21阶段的主要任务/目标包括：1）开发一种 基于鲁棒的四色罗丹明BigDye终止子的单核苷酸延伸 (SNE)阵列分析将允许DNA TaqFS催化的，单核苷酸 延伸以检测与人类相关的多种SNP/突变 癌我们将增加信号强度，并消除对 使用树枝状聚合物包被的聚合物在阵列结合探针上合成昂贵的间隔区 2）扩大试验规模，同时监测30-50 单个阵列上的SNP/突变。多重检测将在PCR和 将检测SNE步骤和总基因组DNA靶标的使用。战略将 还可以实现在单个芯片上筛选多个个体， 相同的突变。R33阶段的目的包括：1）SNE测定法应用于 在lp、llq和14 q中的最小缺失区域内和侧翼的SNP类型， 神经母细胞瘤儿科患者。该测定将有助于鉴定 删除位置和程度，最终将有助于理解 基因型和表型之间的关系;和2）鉴定 突变簇区域内发生的单碱基变化 家族性腺瘤性结肠息肉病（APC）基因 息肉病（FAP）和散发性结直肠癌。我们还将评估 使用SNE突变信息的基因型/表型关系 基于阵列的全基因组mRNA表达谱的分析和结果 已经由NCI合同资助。目标1生成的基于阵列的结果 和2将进行比较，并与以前键入的数据进行对比 个人使用更传统的DNA分析方法，以及 在PE-Biosystems生成的溶液相结果。开发和验证 用于监测癌症中DNA变化的阵列结合方法应该 促进高通量、并行处理样品，用于关键人类 癌症基因此外，它将有助于建立所需的基本知识， 快速确定特定恶性肿瘤的分子基础， 为合理的分子生物学评价提供依据。

DESCRIPTION: (Applicant's Description) Development of cost-effective, high-throughput sampling coupled to rapid, parallel DNA profiling methods will facilitate molecular analysis of normal and diseased states. This R21/R33 initiative focuses on detection of single nucleotide polymorphisms (SNPs) and point mutations in cancer. Our goals are as follows: Milestones/Aims for the R21 phase include: 1) Development of a robust, 4-color rhodamine BigDye terminators-based single nucleotide extension (SNE) array assay which will allow DNA TaqFS-catalyzed, single nucleotide extension to detect a wide variety of SNPs/mutations associated with human cancer. We will increase signal intensity as well as eliminate the need for synthesis of costly spacers on array-bound probes by using dendrimer-coated surfaces; and, 2) Scale-up the assay to simultaneously monitor 30-50 SNPs/mutations on a single array. Multiplexing will be done both at PCR and SNE steps and use of total genomic DNA targets will be tested. Strategies will also be implemented to screen multiple individuals on a single chip for the same mutation. Aims for the R33 phase include: 1) Application of SNE assay to type SNPs within and flanking the minimal deleted region in lp, llq and 14q in neuroblastoma pediatric patients. The assay will facilitate identification of deletion location and extent and eventually will facilitate understanding the relationship between genotype and phenotype; and 2) Identification of single-base changes occurring within the mutation cluster region of adenomatous polyposis coli (APC) gene in patients with familial adenomatous polyposis (FAP) and in sporadic colorectal cancer. We will also evaluate genotype/phenotype relationships using information from SNE-based mutation analyses and results from array-based genome-wide mRNA expression profiles already funded by an NCI Contract. Array-based results generated from Aims 1 and 2 will be compared and contrasted to data available from previously typed individuals using more traditional DNA analysis approaches as well as to solution-phase results generated at PE-Biosystems. Development and validation of array-bound approaches for monitoring DNA changes in cancer should facilitate high throughput, parallel processing of samples for critical human cancer genes. In addition, it will help establish basic knowledge required to rapidly define the molecular basis of specific malignancies and eventually provide a foundation for rational molecular assessment of therapeutic.