DNA microarray surface analysis to optimize detection

描述（由申请人提供）：本生物工程研究组R01竞争延续提案旨在继续阐明商业和模型核酸微阵列诊断测定的关键表面性质的生产力和前一资助期的影响。尽管这些多重诊断形式具有巨大的临床和环境应用潜力，但只有两种微阵列测定法被FDA批准用于临床诊断用途。阵列表面捕获问题妨碍了测定的可靠性、灵敏度和再现性，这些问题发生在所有商业平台上、单个平台中的批次内、不同临床实验室之间、甚至单个载玻片上的微阵列元件内。这也是商业开发中的相当大一部分检测的最重要问题：涉及不同打印技术和检测试剂的定制打印阵列。因此，微阵列的临床影响目前非常有限。该提案的重点是获得理解和解决测定可靠性、灵敏度和重现性问题所需的新信息，这一点具有重要意义。最近FDA、EPA和NIH的疾病、药物药理学和毒性筛选目标的关键路径倡议广泛采用微阵列技术，这依赖于提高测定性能和对其局限性的清晰理解。这项建议直接回应了这一需要。工作的假设是，精确定量的表面固定和捕获效率的微阵列，包括使用新的高分辨率仪器的核酸将确定当前的测定限制，并促进改善表面捕获测定性能所需的临床使用这些诊断。三年计划的具体目标包括：目标1。研究和关联用于核酸分子分析的新的表面分析技术方法，具体地，以量化微阵列表面上的印刷和捕获的核酸密度。目标二。比较DNA和PNA微阵列打印点的均匀性，因为它涉及到打印基板的条件和化学，并解决点到点的变化，在高化学分辨率的微阵列检测方差和商业微阵列基板上观察到的不确定性的共同来源。目标3.通过直接定量和检测复杂介质中的少量靶DNA而无需预纯化，提高标准微阵列检测性能要求和捕获检测检测限。该提案的重点是提高核酸微阵列生物测定的现有能力。一套互补的国家的最先进的表面分析方法将被用来评估影响DNA表面固定和目标杂交模型和商业分析格式的变量。提出的战略解决了这些检测中的重要生物分析问题，以确定影响监测公共卫生和疾病、药理学和个性化医疗以及毒理学的高优先级科学和技术的改进。开发能够以阿托摩尔水平检测来自真实世界样品的DNA杂交的可靠生物测定将允许超出当前研究用途的快速临床诊断应用。促进这些广泛使用的微阵列检测格式从目前的研究到真正的临床未来使用的翻译是本研究的主要目的。

DESCRIPTION (provided by applicant): This Bioengineering Research Group R01 competing continuation proposal aims to continue the productivity and impact of the preceding funded period in elucidating key surface properties of commercial and model nucleic acid microarray diagnostic assays. Despite immense potential clinical and environmental applications for these multiplexed diagnostic formats, only two microarray assays are FDA-approved for clinical diagnostic use. Array surface-capture problems preclude assay reliability, sensitivity, and reproducibility, occurring across all commercial platforms, within batches in a single platform, between different clinical labs, and even within microarray elements on a single slide. This is also the most significant problem for a substantial fraction of assays under commercial development: custom printed arrays involving different printing technologies and assay reagents. Hence, clinical impact of microarrays is currently very limiting. Significance is derived from this proposal's focus on obtaining new information necessary for understanding and resolving the issues of assay reliability, sensitivity and reproducibility. Wide adoption of microarray technology for recent FDA, EPA and NIH Critical Path initiatives for disease, drug pharmacology and toxicitiy screening goals relies on improving assay performance and developing clear understanding of their limitations. This proposal responds directly to that need. The working hypothesis is that precise quantitation of surface immobilization and capture efficiencies in microarrays comprising nucleic acids using newly available high-resolution instrumentation will identify current assay limitations, and facilitate improved surface-capture assay performance necessary for clinical use of these diagnostics. Specific aims for the 3-year project include: Aim 1. Validate and correlate new surface analytical technology methods for molecular analysis of nucleic acids, specifically, to quantify printed and captured nucleic acid density on microarrayed surfaces. Aim 2. Compare DNA and PNA microarray printed spot uniformity as it relates to printed substrate conditions and chemistry, and address spot-to-spot variability at high chemical resolution as common sources of microarray assay variance and uncertainty observed on commercial microarray substrates. Aim 3. Improve standard microarray assay performance requirements and capture assay detection limits by direct quantitation and detection of small amounts of target DNA in complex media without pre-purification. This proposal focuses on improving the current capabilities in nucleic acid microarray bioassay. A suite of complementary state-of-the-art surface analytical methods will be exploited to assess variables affecting DNA surface immobilization and target hybridization in model and commercial assay formats. The strategy proposed addresses important bio-analytical problems in these assays to identify improvements that impact high-priority science and technology involved in monitoring public health and disease, pharmacology and personalized medicine, and toxicology. Developing reliable bio- assays capable of detecting DNA hybridization from real-world samples at an attomole level would allow rapid clinical diagnostic applications beyond current research use. Facilitating translation of these widely used microarray assay formats from current research to true clinical future use is a major aim of this research.