This is a SBIR Fast-Track application to address two currently unmet needs. These are for a quantitative theoretical understanding of DNA multiplex hybridization reactions; and a general design methodology for developing rapid, reliable, multiplex genotyping assays. First, in a broader sense there is presently a void in theoretical understanding of DNA multiplex hybridization. For example, current analytical understanding of the thermodynamics and kinetics of the multiplex microarray hybridization process is minimal. Plans in Phase I include development and implementation of our theoretical multiplex analytical methods, and experimentally demonstrate feasibility of using temperature dependent kinetics to discriminate a perfect match duplex from one containing a single base pair mismatch, i.e. SNP, on a microarray. If feasibility can be established, the first part of Phase II is focused on quantitative evaluation of essential experimental parameters, and further parameterization of the theoretical model. This will establish a solid and more rigorous analytical basis for understanding DNA multiplex hybridization reactions. This theoretical foundation will immediately enable more insightful design, more accurate analysis, and greatly expanded and enhanced predictive capabilities for novel and improved design of microarray based assays. This analytical foundation will also support development and applications of novel diagnostic microarray formats that incorporate and utilize time and temperature as key assay parameters. In the second part of Phase II, to demonstrate capability and immediate utility of our analytical process, we will design, build, test and validate a rapid genotyping panel test for 11 single nucleotide polymorphisms in eight genes that have been associated with Adult Macular Degeneration, a leading cause of age related vision loss. The developed process can be generally applied to the design of multiplex genotyping assays for the detection of virtually any SNP panel for any standard microarray platform. Principal Investigator: Benight, Albert S. Project Narrative The goal of this SBIR Fast-Track project is to develop new and powerful analytical tools that will be parameterized and implemented to provide capabilities of quantitative modeling of the temperature dependence of DNA multiplex hybridization kinetics on a microarrray. The developed tools will be readily applicable to address prescient needs in custom diagnostic assay design. A demonstration of the developed technology will be a rapid and cheap genotyping assay for detection of Single Nucleotide Polymorphisms (SNPs) that may be associated with Adult Macular Degeneration, a genetic disease responsible for nearly 50% of age related vision loss in developed countries. ? ? ?
