DNA Multiplex Hybridization Kinetics and SNP Assays

描述(由申请人提供)：这是一个SBIR快速通道申请，以解决目前未得到满足的两个需求。这些是为了对DNA多重杂交反应进行定量的理论理解，以及开发快速、可靠的多重基因分型分析的一般设计方法。首先，在更广泛的意义上，目前对DNA多重杂交的理论理解是空白的。例如，目前对多重微阵列杂交过程的热力学和动力学的分析了解很少。第一阶段的计划包括开发和实施我们的理论多重分析方法，并通过实验证明使用温度相关动力学在微阵列上区分完全匹配的双链和包含单个碱基对错配的双链(即SNP)的可行性。如果可行，第二阶段的第一部分将集中于对基本实验参数的定量评估，并进一步对理论模型进行参数化。这将为理解DNA多重杂交反应奠定坚实和严谨的分析基础。这一理论基础将立即实现更有洞察力的设计、更准确的分析，并极大地扩展和增强对基于微阵列的分析的新颖和改进设计的预测能力。这一分析基础还将支持新型诊断微阵列格式的开发和应用，这些格式结合并利用时间和温度作为关键的分析参数。在第二阶段的第二部分，为了展示我们的分析过程的能力和立竿见影的效果，我们将设计、构建、测试和验证一个快速基因分型小组测试，测试8个基因中的11个单核苷酸多态，这些基因与成人黄斑变性有关，成人黄斑变性是年龄相关性视力丧失的主要原因。所开发的方法可普遍应用于用于检测任何标准微阵列平台的几乎任何SNP面板的多重基因分型分析的设计。主要研究人员：贝尼特，阿尔伯特·S项目描述这个SBIR快速通道项目的目标是开发新的和强大的分析工具，这些工具将被参数化和实施，以提供在微阵列上对DNA多重杂交动力学的温度依赖关系进行定量建模的能力。开发的工具将很容易适用于解决定制诊断分析设计中的先见之明需求。这项开发的技术的一个示范将是一种快速且廉价的基因分型方法，用于检测可能与成人黄斑变性相关的单核苷酸多态(SNPs)。成人黄斑变性是一种遗传病，在发达国家，近50%的年龄相关性视力丧失是由这种遗传病造成的。

DESCRIPTION (provided by applicant): This is a SBIR Fast-Track application to address two currently unmet needs. These are for a quantitative theoretical understanding of DNA multiplex hybridization reactions; and a general design methodology for developing rapid, reliable, multiplex genotyping assays. First, in a broader sense there is presently a void in theoretical understanding of DNA multiplex hybridization. For example, current analytical understanding of the thermodynamics and kinetics of the multiplex microarray hybridization process is minimal. Plans in Phase I include development and implementation of our theoretical multiplex analytical methods, and experimentally demonstrate feasibility of using temperature dependent kinetics to discriminate a perfect match duplex from one containing a single base pair mismatch, i.e. SNP, on a microarray. If feasibility can be established, the first part of Phase II is focused on quantitative evaluation of essential experimental parameters, and further parameterization of the theoretical model. This will establish a solid and more rigorous analytical basis for understanding DNA multiplex hybridization reactions. This theoretical foundation will immediately enable more insightful design, more accurate analysis, and greatly expanded and enhanced predictive capabilities for novel and improved design of microarray based assays. This analytical foundation will also support development and applications of novel diagnostic microarray formats that incorporate and utilize time and temperature as key assay parameters. In the second part of Phase II, to demonstrate capability and immediate utility of our analytical process, we will design, build, test and validate a rapid genotyping panel test for 11 single nucleotide polymorphisms in eight genes that have been associated with Adult Macular Degeneration, a leading cause of age related vision loss. The developed process can be generally applied to the design of multiplex genotyping assays for the detection of virtually any SNP panel for any standard microarray platform. Principal Investigator: Benight, Albert S. Project Narrative The goal of this SBIR Fast-Track project is to develop new and powerful analytical tools that will be parameterized and implemented to provide capabilities of quantitative modeling of the temperature dependence of DNA multiplex hybridization kinetics on a microarrray. The developed tools will be readily applicable to address prescient needs in custom diagnostic assay design. A demonstration of the developed technology will be a rapid and cheap genotyping assay for detection of Single Nucleotide Polymorphisms (SNPs) that may be associated with Adult Macular Degeneration, a genetic disease responsible for nearly 50% of age related vision loss in developed countries.