The goal of this proposal is to develop a single step method for the parallel scoring of single nucleotide polymorphisms (SNPs) in a surface array format. The approach is based upon a recently developed invasive cleavage assay for SNP scoring referred to as the 'Invader"""""""" assay (Third Wave Technologies, Inc.) As it is a signal amplification technology rather than a target amplification technology, this assay is not subject to the contamination/carryover problems characteristic of PCR. It is a very simple, robust, and isothermal assay well-suited for high throughput analysis. In the proposed surface-based version of the procedure (the """"""""Surface Invader Assay""""""""), addition to the surface of target human genomic DNA containing a given SNP allele will result in specific cleavage of a corresponding surface-immobilized probe oligonucleotide containing a fluorophore-quencher dye pair. This cleavage will occur between the fluorophore and the quencher, leaving the unquenched fluorophore attached to the surface. The fluorescence intensity of this unquenched fluorophore will be substantially greater than that of the quenched fluorophore, and thus the region of the DNA array containing that probe oligonucleotide will exhibit increased fluorescence intensity in a specific target-dependent fashion. By employing an array of such probe oligonucleotides, one for each SNP allele to be typed, a single addition of target human genomic DNA to the surface, along with the other needed assay reagents (buffer and enzyme), followed by incubation, washing, and fluorescence imaging steps, will yield the genotypes of the target DNA analyzed for each SNP represented in the array.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG002298-02
Application #
6536485
Study Section
Genome Study Section (GNM)
Program Officer
Ozenberger, Bradley
Project Start
2001-06-15
Project End
2004-05-31
Budget Start
2002-06-01
Budget End
2003-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$363,750
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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