Development of Fluorescent OFRG Technology

Approach: OFRG involves the array of microbial small subunit rRNa genes onto membranes followed by hybridization with radioactive 10-nucleotide probes. Depending on technology used, 1,500 to 10,000 genes may be analyzed simultaneously in this manner. Fluorescent labeling of the probes shows promise but has been unsuccessful to date because fluorophore size interferes with hybridization. Possible probe sets are generated using simulated annealing and Lagrangian relaxation algorithms, and probes are tested empirically to generate final sets. Probe sets, various fluorophores and fluorophore-spacer combinations will be tested to determine chemistries that will allow probe-target binding. Fluorescent probe sets for bacteria and fungi are proposed. Development of these probe sets will not only allow us to remove the need for using radioactive materials, but it will also allow analysis of up to approximately 100,000 genes simultaneously, which would dramatically increase our experimental capabilities.