DNA Microarray Surface Analysis to Optimize Detection

DNA 微阵列表面分析以优化检测

• 批准号: 6772428
• 负责人: DAVID W GRAINGER
• 金额: $ 35.19万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-15 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6772428
• 关键词: DNA; enzyme linked immunosorbent assay; gene expression; microarray technology; nucleic acid probes; nucleic acid quantitation /detection; polymerase chain reaction; spectrometry; tissue /cell culture

DESCRIPTION (provided by applicant): Microarray-based information, now routine in most medical research communities, pervades most aspects of diagnostics, sensing, biotechnology, oncology and pathology. Detection limits and selectivities for DNA targets are far below theoretical performance limits, but very little information is reported or known about the chemical, physical or biological fate of full length DNA, cDNA or oligo-DNA probes immobilized on any of the diverse set of microarray surfaces. How immobilized DNA surface disposition influences subsequent hybridization efficiency, and reliability of array data interpretation and assay quantification is unknown. Quantitative interpretation of DNA microarray signal intensity is currently very difficult since factors influencing DNA probe-target interactions at microarray surfaces have not been analyzed with high-resolution surface analytical methods often applied to other biomedical surface problems. Our hypothesis is that DNA microarray target hybridization efficiency and diagnostic target detection limits in biomedical samples are correlated directly with the orientation, density, and immobilization efficiency of probe DNA on microarray surfaces. To investigate this hypothesis, we propose the following Specific Aims: 1. Establish a quantitative understanding of the correlation between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples using radiometric 32P-DNA assay and optical imaging on several surface chemistry platforms and assess reliability and reproducibility issues in these strategies; 2. Develop reliable, quantitative methods for high-resolution surface analysis of DNA density and orientational populations on ss-cDNA and hybridized ds-DNA on arraying surfaces using modem biomedically relevant methods (XPS, ToF-SIMS and optical anisotropy of immobilized DNA). The overall objective is to correlate high-resolution surface analytical data on DNA arrays with radiometric measurements to establish a non-radiometric 'standard curve' to assess DNA immobilization on microarray surfaces more conveniently and accurately. Moreover, orientational information on immobilized DNA using a combination of innovative spectroscopy methods will be correlated to immobilized DNA density and hybridization efficiency in array formats. All methods will converge to produce a fundamental understanding of microarray surface & hybridization performance limitations currently not available.

描述（由申请人提供）： 基于微阵列的信息现在已成为大多数医学研究领域的常规信息，并渗透到诊断、传感、生物技术、肿瘤学和病理学的大多数方面。 DNA 靶标的检测限和选择性远低于理论性能极限，但关于固定在任何不同微阵列上的全长 DNA、cDNA 或寡聚 DNA 探针的化学、物理或生物命运，报道或了解的信息很少表面。固定化 DNA 表面处置如何影响随后的杂交效率以及阵列数据解释和测定定量的可靠性尚不清楚。 DNA 微阵列信号强度的定量解释目前非常困难，因为尚未使用通常应用于其他生物医学表面问题的高分辨率表面分析方法来分析影响微阵列表面的 DNA 探针-靶相互作用的因素。我们的假设是，生物医学样品中 DNA 微阵列靶标杂交效率和诊断靶标检测限与微阵列表面上探针 DNA 的方向、密度和固定效率直接相关。为了研究这一假设，我们提出以下具体目标： 1. 在多个表面化学平台上使用放射 32P-DNA 测定和光学成像，定量了解微阵列表面固定探针 DNA 密度与生物样品中靶标杂交效率之间的相关性并评估这些策略的可靠性和可重复性问题； 2. 使用现代生物医学相关方法（XPS、ToF-SIMS 和固定 DNA 的光学各向异性）开发可靠的定量方法，对阵列表面上 ss-cDNA 和杂交 ds-DNA 的 DNA 密度和定向群体进行高分辨率表面分析。总体目标是将 DNA 阵列上的高分辨率表面分析数据与辐射测量相关联，以建立非辐射“标准曲线”，从而更方便、更准确地评估微阵列表面上的 DNA 固定。此外，使用创新光谱方法组合的固定化 DNA 的方向信息将与阵列格式中的固定化 DNA 密度和杂交效率相关。所有方法将汇聚在一起，以产生对目前尚不可用的微阵列表面和杂交性能限制的基本了解。