DNA Microarray Surface Analysis to Optimize Detection

描述（由申请人提供）： 基于微阵列的信息，现在在大多数医学研究社区中是常规的，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靶的检测极限和选择性远低于理论性能极限，但关于固定在任何不同的微阵列表面上的全长DNA、cDNA或寡聚DNA探针的化学、物理或生物命运的信息报道或已知很少。固定化DNA表面配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是未知的。DNA微阵列信号强度的定量解释是目前非常困难的，因为影响DNA探针-靶相互作用的因素在微阵列表面没有分析与高分辨率的表面分析方法，往往适用于其他生物医学表面问题。我们的假设是，生物医学样本中DNA微阵列靶点杂交效率和诊断靶点检测限与微阵列表面探针DNA的方向、密度和固定效率直接相关。为了研究这一假设，我们提出了以下具体目标：1。在几个表面化学平台上使用放射性32 P-DNA测定和光学成像建立对微阵列表面上的固定化探针DNA密度与生物样品中的靶杂交效率之间的相关性的定量理解，并评估这些策略中的可靠性和再现性问题; 2.使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），开发可靠的定量方法，用于阵列表面上ss-cDNA和杂交ds-DNA上DNA密度和取向群体的高分辨率表面分析。总体目标是将DNA阵列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，使用创新的光谱学方法的组合的固定化DNA上的取向信息将相关的固定化DNA的密度和杂交效率的阵列格式。所有的方法将收敛到产生一个基本的理解微阵列表面和杂交性能的限制，目前还没有。

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.