FOUR COLOR ARRAY BOUND SNP/MUTATION DETECTION IN CANCER

描述：(申请人描述) 开发具有成本效益的高通量采样与快速、 并行DNA图谱分析方法将促进正常 以及疾病缠身的州。这项R21/R33计划侧重于检测单个 癌症中的核苷酸多态(SNPs)和点突变。我们的目标是 R21阶段的里程碑/目标包括：1)制定 基于强健的四色罗丹明大染料终止子的单核苷酸延伸 (SNE)阵列检测将允许DNA TaqFS催化的单核苷酸 扩展以检测与人类相关的多种SNPs/突变 癌症。我们将增加信号强度，同时消除 用树枝状大分子包覆合成阵列结合探针上昂贵的间隔物 表面；以及，2)放大化验以同时监测30-50 单个阵列上的SNPs/突变。多路传输将在聚合酶链式反应和 SNE步骤和总基因组DNA靶标的使用将进行测试。战略将 还可以在单个芯片上对多个人进行筛查 同样的突变。R33阶段的目标包括：1)SNE测定在 在Lp、L1q和14q中最小缺失区域内和两侧键入SNP 儿童神经母细胞瘤患者。该化验将有助于鉴定 删除的位置和范围，最终将有助于理解 基因型与表型的关系；2)鉴定 发生在突变簇区内的单碱基变化 家族性腺瘤患者的腺瘤性息肉病结肠(APC)基因 息肉病(FAP)和散发性结直肠癌。我们还将评估 使用基于SNE的突变信息的基因型/表型关系 基于阵列的全基因组mRNA表达谱的分析和结果 已经得到了NCI合同的资助。AIMS 1生成的基于数组的结果 和2将与以前输入的数据进行比较和对比 使用更传统的DNA分析方法的个人以及 PE-BiosSystems产生的溶液阶段结果。开发和验证 用于监测癌症中DNA变化的阵列结合方法应该 促进关键人类样本的高吞吐量、并行处理 此外，它还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，并最终 为合理进行治疗性分子评价提供了依据。

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.