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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB001473-03
Application #
6888132
Study Section
Special Emphasis Panel (ZRG1-SSS-W (02))
Program Officer
Korte, Brenda
Project Start
2003-07-15
Project End
2007-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$362,477
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Rao, Archana N; Grainger, David W (2014) BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE. Biomater Sci 2:436-471
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Harrison, Andrew; Binder, Hans; Buhot, Arnaud et al. (2013) Physico-chemical foundations underpinning microarray and next-generation sequencing experiments. Nucleic Acids Res 41:2779-96
Rao, Archana N; Rodesch, Christopher K; Grainger, David W (2012) Real-time fluorescent image analysis of DNA spot hybridization kinetics to assess microarray spot heterogeneity. Anal Chem 84:9379-87
Graham, Daniel J; Castner, David G (2012) Multivariate analysis of ToF-SIMS data from multicomponent systems: the why, when, and how. Biointerphases 7:49
Rao, Archana N; Vandencasteele, Nicolas; Gamble, Lara J et al. (2012) High-resolution epifluorescence and time-of-flight secondary ion mass spectrometry chemical imaging comparisons of single DNA microarray spots. Anal Chem 84:10628-36
Dubey, Manish; Brison, J; Grainger, David W et al. (2011) Comparison of Bi(1), Bi(3) and C(60) primary ion sources for ToF-SIMS imaging of patterned protein samples. Surf Interface Anal 43:261-264
Liu, Fang; Dubey, Manish; Takahashi, Hironobu et al. (2010) Immobilized antibody orientation analysis using secondary ion mass spectrometry and fluorescence imaging of affinity-generated patterns. Anal Chem 82:2947-58
Dubey, Manish; Emoto, Kazunori; Cheng, Fang et al. (2009) Surface Analysis of Photolithographic Patterns using ToF-SIMS and PCA. Surf Interface Anal 41:645-652
Grainger, David W; Castner, David G; Dubey, Manish et al. (2009) Affinity-based Protein Surface Pattern Formation by Ligand Self-Selection from Mixed Protein Solutions. Adv Funct Mater 19:3046-3055

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