DNA Multiplex Hybridization Kinetics and SNP Assays

描述（由申请人提供）：这是SBIR快速通道申请，以解决两个目前未满足的需求。这些是对DNA多重杂交反应的定量理论理解；以及开发快速、可靠、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，目前在DNA多重杂交的理论认识上是一个空白。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理解是最小的。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依赖动力学在微阵列上区分完美匹配双工和包含单个碱基对不匹配的双工（即SNP）的可行性。如果能够确定可行性，第二期的第一部分将重点对关键实验参数进行定量评价，并进一步对理论模型进行参数化。这将为理解DNA多重杂交反应建立坚实而严谨的分析基础。这一理论基础将立即使更有洞察力的设计，更准确的分析，并大大扩展和增强了基于微阵列的分析的新颖和改进设计的预测能力。该分析基础还将支持新型诊断微阵列格式的开发和应用，该格式将时间和温度作为关键分析参数。在II期的第二部分，为了证明我们的分析过程的能力和直接效用，我们将设计、构建、测试和验证一个快速基因分型面板测试，用于检测与成人黄斑变性相关的8个基因中的11个单核苷酸多态性，成人黄斑变性是与年龄相关的视力丧失的主要原因。开发的过程可以普遍应用于多重基因分型分析的设计，用于检测几乎任何标准微阵列平台的任何SNP面板。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并实施，以提供在微阵列上DNA多重杂交动力学的温度依赖性的定量建模能力。开发的工具将很容易适用于解决定制诊断分析设计的先见之明需求。该技术的示范将是一种快速、廉价的基因分型检测方法，用于检测可能与成人黄斑变性相关的单核苷酸多态性（snp）。在发达国家，成人黄斑变性是一种遗传性疾病，导致近50%的年龄相关性视力丧失。

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.