In this competitive renewal proposal we seek to continue the development of """"""""surface invader"""""""" DNA arrays for high throughput SNP analysis. During the prior grant period we developed the surface invader assay and demonstrated """"""""proof-of-principle"""""""" for the parallel analysis of SNPs directly from unamplified human genomic DNA samples on spotted DNA arrays. Now we desire to take this technology to the next step, i.e. to implement this chemistry in a high-density DNA array format, where the DNA array is manufactured by photolithographic methods. There are three issues that will be addressed to enable fabrication of the needed DNA arrays. First, chemistry will be implemented for the photolithographic synthesis of DNA molecules in the 5'->3' direction. Second, chemistry will be developed and implemented to allow the photolithographic synthesis of two different sequences intermixed in a single array element; both are needed to form the / necessary ternary complex on the surface. Third, glassy carbon and/or diamond thin film substrates will be developed for use in photolithographic DNA synthesis. A secondary thrust of this proposal will be to investigate an approach to detection based upon the detection of single molecule cleavage events on the surface. The rolling circle amplification reaction can make DNA molecules hundreds of thousands of bases long from a single initiation site. As it is very easy to detect such long DNA molecules, this raises the possibility of directly detecting individual cleaved DNA molecules on the surface; in fact, our preliminary results have shown this capability. This is intriguing, as it opens the possibility of analyzing very low levels of nucleic acid targets, perhaps as little as a single molecule, with many applications in areas such as gene expression analysis, infectious disease diagnostics, biowarfare agent detection, and genotyping from minute samples such as buccal swabs or fingerprick blood samples.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG002298-05
Application #
7072792
Study Section
Special Emphasis Panel (ZRG1-ISD (01))
Program Officer
Ozenberger, Bradley
Project Start
2001-06-15
Project End
2008-05-31
Budget Start
2006-06-01
Budget End
2007-05-31
Support Year
5
Fiscal Year
2006
Total Cost
$581,129
Indirect Cost
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Lockett, Matthew R; Smith, Lloyd M (2015) Carbon Substrates: A Stable Foundation for Biomolecular Arrays. Annu Rev Anal Chem (Palo Alto Calif) 8:263-85
Mandir, Joshua B; Lockett, Matthew R; Phillips, Margaret F et al. (2009) Rapid determination of RNA accessible sites by surface plasmon resonance detection of hybridization to DNA arrays. Anal Chem 81:8949-56
Lockett, Matthew R; Carlisle, Justin C; Le, Dinh V et al. (2009) Acyl chloride-modified amorphous carbon substrates for the attachment of alcohol-, thiol-, and amine-containing molecules. Langmuir 25:5120-6
Chen, Siyuan; Smith, Lloyd M (2009) Photopatterned thiol surfaces for biomolecule immobilization. Langmuir 25:12275-82
Lockett, Matthew R; Smith, Lloyd M (2009) Fabrication and characterization of DNA arrays prepared on carbon-on-metal substrates. Anal Chem 81:6429-37
Chen, Siyuan; Phillips, Margaret F; Cerrina, Franco et al. (2009) Controlling oligonucleotide surface density in light-directed DNA array fabrication. Langmuir 25:6570-5
Lockett, Matthew R; Smith, Lloyd M (2009) Attaching molecules to chlorinated and brominated amorphous carbon substrates via Grignard reactions. Langmuir 25:3340-3
Lockett, Matthew R; Shortreed, Michael R; Smith, Lloyd M (2008) Aldehyde-terminated amorphous carbon substrates for the fabrication of biomolecule arrays. Langmuir 24:9198-203
Lockett, Matthew R; Weibel, Stephen C; Phillips, Margaret F et al. (2008) Carbon-on-metal films for surface plasmon resonance detection of DNA arrays. J Am Chem Soc 130:8611-3
Phillips, Margaret F; Lockett, Matthew R; Rodesch, Matthew J et al. (2008) In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication. Nucleic Acids Res 36:e7

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