POLYTECHNIC INSTITUTE OF NEW YORK UNIVERSITY

This American Recovery and Reinvestment Act (ARRA) supplement seeks to establish an instrumentation facility to accelerate development of morpholino (MO) microarray technology pursued under the parent award. The proposed instrument will document history of the microarray pattern as it evolves during hybridization, and in this way provide essential information for assessing microarray performance. This request contributes to the US economy in following ways: by streamlining validation of MO technology for nucleic acid analysis, the request advances domestic innovation in high-throughput analytical genomics; by demonstrating US startup technology for real-time microarray imaging, the request further develops domestic hardware for biodiagnostic applications; by providing 6 months of postdoctoral support to establish real-time microarray protocols, the request invests in human resources and job creation. The requested instrument is based on a total-internal-reflection geometry for excitation of fluorescence; in this TIRF geometry, excitation radiation is confined to the near-surface region so that background from fluorescent species in solution is suppressed. Low solution background enables in-situ, independent monitoring of hybridization which, combined with high performance imaging hardware, allows the hybridization process to be simultaneously tracked at each microarray feature or "spot". From this real-time data, rates of hybridization, cross hybridization, and wash-off of mismatched sequences can be determined, and realization of equilibrium states can be confirmed. This expanded information will expedite validation of MO microarrays by assessing their performance relative to DNA microarray technology, as well as facilitate optimization of hybridization protocols. TIRF imaging will thus remove a principal bottleneck in validation and fundamental studies of MO microarray technology pursued under the parent award.

Clarification of Codes