HIGH THROUGHPUT SNP DISCOVERY & SCORING

高通量 SNP 发现

• 批准号: 7724226
• 负责人: JOHN P NOLAN
• 金额: $ 3.22万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724226
• 关键词: Address; Binding; Binding Proteins; Biological Assay; Chromosome Mapping; Clinical; Collaborations; Compatible; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Sequence Analysis; Diagnosis; Diagnostic; Disease; Disease susceptibility; Dyes; Flow Cytometry; Funding; Genes; Genetic Variation; Genomics; Goals; Grant; Heteroduplex DNA; Immobilization; Institution; Label; Laboratories; Measurement; Methods; Microspheres; Pharmacogenetics; Polymerase; Polymorphism Analysis; Rate; Research; Research Institute; Research Personnel; Resources; Sampling; Scanning; Scheme; Score; Scoring Method; Single Nucleotide Polymorphism; Source; Technology; United States National Institutes of Health; Universities; Variant; base; day; dideoxynucleotide; novel strategies

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Analysis of DNA sequence variation is important for identifying disease related genes and diagnosing disease susceptibility. The most common type of genetic variation is the single nucleotide polymorphism (SNP), which may occur as frequently as 1 in every 1000 DNA bases. The goal of this project is to develop, validate, and demonstrate new approaches to the discovery and scoring of single nucleotide polymorphisms (SNPs). The new methods will take advantage of versatile and widely available measurement platform, flow cytometry, to provide rapid and sensitive sample analysis without wash steps. De novo SNP detection will be achieved using an immobilized mismatch binding protein to bind fluorescently labeled heteroduplex DNA to microspheres, which will then be analyzed by flow cytometry. We will evaluate different mismatch binding proteins, immobilization approaches, and labeling strategies to develop a homogeneous assay compatible with multiplexed analysis by flow cytometry. The optimized assay will be sensitive, rapid, and scaleable to scan amplified genomic sequence for SNPs at rates of greater than 1 Mb/day. SNP scoring will be performed using microsphere-based minisequencing in which primers will be extended with fluorescent dideoxynucleotides using polymerase, and then captured on beads and analyzed by flow cytometry. By employing a unique scheme of capture sequences to address differently dyed microspheres, we will be able to score simultaneously dozens, and potentially hundreds, of SNPs from a single genomic sample in 1-2 minutes. We will validate and demonstrate these new approaches in collaboration with laboratories currently using conventional technologies to discover and score SNPs for use in disease diagnostics, pharmacogenetics, and mapping and linkage studies. Both our SNP discovery and scoring methods will be compatible commercial flow cytometers present in most universities, research institutes, and clinical diagnostic laboratories.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 DNA序列变异的分析对于鉴定疾病相关的基因和诊断疾病易感性很重要。 遗传变异的最常见类型是单核苷酸多态性（SNP），每1000个DNA碱基中的发生频率约为1。 该项目的目的是开发，验证和展示单核苷酸多态性（SNP）的发现和评分的新方法。 新方法将利用多功能且可广泛可用的测量平台流式细胞仪，以提供无需洗涤步骤的快速和敏感的样品分析。将使用固定的错配结合蛋白来实现从头SNP检测，以结合荧光标记的异源性DNA与微球，然后通过流式细胞仪对其进行分析。 我们将评估不同的不匹配结合蛋白，固定方法和标记策略，以开发出与流式细胞仪多重分析兼容的均匀测定。 优化的测定将对SNP的扫描放大基因组序列敏感，快速且可扩展，以大于1 MB/天的速率。 SNP评分将使用基于微球的微型定量进行，其中将使用聚合酶使用荧光二氧核苷酸扩展引物，然后在珠子上捕获并通过流式细胞仪进行分析。 通过采用独特的捕获序列方案来解决不同染色的微球，我们将能够在1-2分钟内同时从单个基因组样本中同时评分数百个SNP。 我们将通过与当前使用常规技术的实验室合作验证和证明这些新方法，以发现和评分SNP，以用于疾病诊断，药物遗传学以及映射和链接研究。 我们的SNP发现和评分方法都将是大多数大学，研究机构和临床诊断实验室中兼容的商业流式细胞仪。