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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 DNA序列变异分析对于识别疾病相关基因和诊断疾病易感性具有重要意义。 最常见的遗传变异类型是单核苷酸多态性 (SNP)，其出现频率可能为每 1000 个 DNA 碱基中就有 1 个。 该项目的目标是开发、验证和演示单核苷酸多态性 (SNP) 的发现和评分的新方法。 新方法将利用通用且广泛使用的测量平台流式细胞术，提供快速、灵敏的样品分析，无需清洗步骤。使用固定的错配结合蛋白将荧光标记的异源双链 DNA 与微球结合，从而实现从头 SNP 检测，然后通过流式细胞术进行分析。 我们将评估不同的错配结合蛋白、固定方法和标记策略，以开发与流式细胞术多重分析兼容的均相测定。 优化后的检测方法灵敏、快速且可扩展，能够以大于 1 Mb/天的速度扫描扩增的基因组序列中的 SNP。 SNP 评分将使用基于微球的小测序进行，其中使用聚合酶用荧光双脱氧核苷酸延伸引物，然后捕获在珠子上并通过流式细胞术进行分析。 通过采用独特的捕获序列方案来处理不同染色的微球，我们将能够在 1-2 分钟内从单个基因组样本中同时对数十个甚至可能数百个 SNP 进行评分。 我们将与目前使用传统技术的实验室合作验证和演示这些新方法，以发现和评分 SNP，以用于疾病诊断、药物遗传学以及绘图和连锁研究。 我们的 SNP 发现和评分方法都将与大多数大学、研究机构和临床诊断实验室中使用的商业流式细胞仪兼容。