DNA Microarray Surface Analysis to Optimize Detection

描述(由申请人提供)： 基于微阵列的信息现在是大多数医学研究社区的常规信息，覆盖了诊断学、传感、生物技术、肿瘤学和病理学的大多数方面。DNA靶标的检测下限和选择性远低于理论性能下限，但关于固定在各种不同微阵列表面的全长DNA、cDNA或寡核苷酸探针的化学、物理或生物命运的信息报道或知道的很少。固定的DNA表面处理如何影响随后的杂交效率，以及阵列数据解释和分析定量的可靠性尚不清楚。目前，DNA微阵列信号强度的定量解释是非常困难的，因为影响微阵列表面DNA探针-靶相互作用的因素还没有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。我们的假设是，DNA微阵列靶标杂交效率和生物医学样本中诊断靶标的检测下限与探针DNA在微阵列表面的取向、密度和固定效率直接相关。为了研究这一假设，我们提出了以下具体目标：1.利用放射性32P-DNA分析和几个表面化学平台上的光学成像，建立生物样品中固定探针DNA密度与靶向杂交效率之间的定量理解，并评估这些策略的可靠性和重复性问题；2.利用现代生物医学相关方法(XPS、ToF-SIMS和固定化DNA的光学各向异性)，建立可靠、定量的高分辨率表面分析方法，用于高分辨率表面分析ss-cDNA和杂交的ds-DNA表面的DNA密度和定向群体。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，以建立一个非辐射“标准曲线”，以更方便和准确地评估DNA在微阵列表面的固定。此外，结合创新的光谱方法，固定化DNA的定位信息将与固定化DNA密度和阵列形式的杂交效率相关联。所有的方法都将融合在一起，以产生对微阵列表面的基本理解&杂交性能的限制目前是不可用的。

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.