Real-Time Heteroplasmy Analysis on Microarrays

描述(申请人提供)：许多人类疾病--如果不是所有的人类疾病--和人类健康似乎与我们的基因有关。政府和私营部门的主要研究工作都集中在调查这种联系上。不幸的是，对于这项关键工作，我们最好的工具之一--即微阵列--阻碍了我们，因为大多数工具只能提供一般的定性结果，限制了它们的有效性。一个关键的例子是单核苷酸多态(SNP)检测。目前的SNP分析不是定量的，因为不完善的分子识别(交叉杂交)和使用微阵列进行的假平衡分析限制了它们的使用(例如，用于初步筛选，如Affymetrix SNPChip)。微阵列技术试图通过过度冗余来补偿，导致大量不准确/不可重现的数据。对这些海量数据进行分类的需要延长了分析时间，导致不正确的结论，引发科学争议，并可能误导诊断和药物研究。幸运的是，这些成果是可以改进的--这是这里提出的多阶段STTR快速通道项目的主要目标。需要下一代工具进行实时、可靠、定量的SNPs分析的一个关键任务是异质性(半定量线粒体DNA SNP分析)，其中包括新的数据管理/分析能力和简单但强大的化学分析。异质性可以满足对涉及癌症、糖尿病、阿尔茨海默病、高血压和各种神经肌肉、神经退行性疾病和代谢性疾病的可靠/高性价比定量测试的主要需求。我们的目标是基于犹他州大学授权的、由Sigma创始人开发的技术，开发实时和定量异质性研究工具，并将其商业化。使用我们专有的“竞争置换分析”(CDA)--关键创新--的初步数据有力地表明了在这个快速通道项目下成功开发和快速商业化的潜力。具体地说，我们建议开发我们的新方法来执行实时SNP微阵列分析(通过CDA)，方法是在存在未标记靶标的情况下监测已知竞争对手的非线性结合动力学。我们的第一阶段目标是1)展示相关的结合动力学；2)证明/验证在模型系统中使用CDA进行基于异质性的SNP检测的可行性；以及3)基于合成目标、竞争对手和低亲和力物种(背景)，开发基于CDA的多组分模型系统的分析方法。达到关键的第一阶段里程碑将使我们能够实现第二阶段的目标：4)表征CDA以用于A3243G突变基因的异质性分析；5)扩大CDA以询问多个突变；以及6)完成CDA与参考方法的比较验证。第二阶段的成功将提供吸引“第三阶段”工业和金融合作伙伴所需的数据。这将有助于将产品快速推向一个价值数十亿美元的研究/诊断行业的关键利基市场，使全球人类健康受益。

DESCRIPTION (provided by applicant): Many human diseases-if not all human diseases-and human health appear to be linked to our genetics. Major government and private research efforts are focused on investigating this linkage. Unfortunately, one of our best tools for this critical work-i.e., microarrays-holds us back, as most provide only generally qualitative results, limiting their usefulness. One key example is single nucleotide polymorphism (SNP) detection. Current SNP analysis is not quantitative because of imperfect molecular recognition (cross-hybridization) and pseudoequilibrium analyses performed with microarrays, limiting their use (e.g., to preliminary screening, as with the Affymetrix SNPChip). The microarray techniques attempt to compensate via excessive redundancy, leading to massive quantities of inaccurate/irreproducible data. The need to sort through these copious amounts of data extends analysis time, leads to improper conclusions, initiates scientific controversy, and may misdirect diagnostic and pharmaceutical research. Fortunately, these outcomes can be improved upon-which is the primary goal of the multi-phase STTR Fast-Track project proposed here. One key task that requires next-generation tools for real-time, reliable, quantitative SNPs analysis-including new data-management/analysis capabilities and simple yet powerful chemistries-is heteroplasmy (semiquantitative mitochondrial DNA SNP assays). Heteroplasmy could fulfill a major unmet need for reliable/costefficient quantitative tests involving cancer, diabetes, Alzheimer's disease, hypertension and a variety of neuromuscular, neurodegenerative, and metabolic diseases. Our goal is to develop, prototype, and commercialize real-time and quantitative heteroplasmy research tools based on technology licensed from the U of Utah and developed by Sigma founders. Preliminary data using our proprietary "competitive displacement analysis" (CDA)-the key innovation-strongly indicates the potential for successful development and rapid commercialization under this Fast-Track project. Specifically, we propose to develop our new method of performing real-time SNP microarray analysis (via CDA) by monitoring non-linear binding kinetics of known competitors in the presence of unlabeled targets. Our Phase I Aims are to 1) Demonstrate relevant binding kinetics; 2) Prove/validate feasibility of using CDA for heteroplasmy-based SNP detection in a model system; and 3) Develop CDA-based analytical approaches for multi-component model systems, based on synthetic targets, competitors, and lower-affinity species (background). Meeting the key Phase I milestones will allow us to pursue Phase II Aims: 4) Characterize CDA for heteroplasmy analysis on A3243G mutation locus; 5) Scale up CDA to interrogate multiple mutations; and 6) Complete comparative validation of CDA versus reference methods. Phase II success will provide the data needed to attract "Phase III" industry and financial partners. This will facilitate rapid product introduction into a key niche in a multi-billion-dollar research/diagnostics industry that benefits human health worldwide.