HIGH THROUGHPUT SNP DISCOVERY & SCORING

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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(SNP)，其发生频率可能高达每1000个DNA碱基中有1个。这个项目的目标是开发、验证和展示发现和评分单核苷酸多态(SNPs)的新方法。新方法将利用通用和广泛使用的测量平台--流式细胞术，提供快速和灵敏的样品分析，而不需要洗涤步骤。利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，然后用流式细胞仪进行分析，从而实现从头开始的SNP检测。我们将评估不同的错配结合蛋白、固定化方法和标记策略，以开发一种与流式细胞术多重分析兼容的均质分析方法。优化后的方法将是灵敏、快速和可扩展的，以超过1Mb/天的速度扫描扩增的基因组序列以检测SNPs。SNP评分将使用基于微球的微序列进行，其中的引物将使用聚合酶与荧光双脱氧核苷酸延伸，然后在珠子上捕获并通过流式细胞仪进行分析。通过使用一种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获得数十个甚至数百个SNP。我们将与目前使用常规技术的实验室合作，验证和演示这些新方法，以发现和记录用于疾病诊断、药物遗传学以及地图绘制和连锁研究的SNPs。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.