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.
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