HIGH THROUGHPUT SNP DISCOVERY & SCORING

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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疾病易感性诊断具有重要意义。 最常见的遗传变异类型是单核苷酸多态性（SNP），其发生频率可能为每1000个DNA碱基中有1个。 该项目的目标是开发、验证和展示发现和评分单核苷酸多态性（SNP）的新方法。 新方法将利用通用且广泛可用的测量平台流式细胞术，提供快速且灵敏的样品分析，而无需洗涤步骤。使用固定的错配结合蛋白将荧光标记的异源双链DNA结合到微球上，然后通过流式细胞术进行分析，实现从头SNP检测。 我们将评估不同的错配结合蛋白，固定方法和标记策略，以开发一种与流式细胞术多重分析兼容的均相检测方法。 优化的测定将是灵敏的、快速的和可缩放的，以大于1 Mb/天的速率扫描扩增的基因组序列的SNP。 SNP评分将使用基于微球的微测序进行，其中引物将使用聚合酶用荧光双脱氧核苷酸延伸，然后在珠上捕获并通过流式细胞术分析。 通过采用捕获序列的独特方案来处理不同染色的微球，我们将能够在1-2分钟内同时对来自单个基因组样品的数十个，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，验证和展示这些新方法，以发现和评分SNP，用于疾病诊断，药物遗传学以及作图和连锁研究。 我们的SNP发现和评分方法将与大多数大学、研究机构和临床诊断实验室中存在的商用流式细胞仪兼容。

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.