RNA-Based Microbial Community Structure Determination

70524 In order to predict the bioremediation rate and ecological impact of contaminated sites, a rapid method to determine the microbial community structure is needed. Current approaches are time consuming, labor intensive, and expensive. Microarray gene probe technology exhibits extraordinary potential; however, difficulties specific to the application of this technology to ribosomal gene sequences has retarded advancement. This project will develop a two-step process in which a thermal-cycling program to label target recognition oligodeoxynucleotides (trODNs) by repeated single-base extension reactions is followed by a process for the resolution of the labeled reaction products (by competitive hybridization to an array of immobilized probes). Phase I will demonstrate that the trODNs can be selectively labeled using single-base extension reactions. The extent of probe labeling will be shown to be directly related to the number of template molecules present in the labeling reaction. It also will be shown that the technique will reduce problems associated with ribosomal nucleic acid secondary structures, which make the quantitative description of microbial community structure based on ribosomal sequences intractable. Commercial Applications and Other Benefits as described by the awardee: The technology should serve as an essential component of environmental microbial community structure analysis, contributing to our understanding of the general ecology of microorganisms in contaminated sites, subsurface soils and sediments, gastrointestinal tracts, and other environments. The principle could also be extended to pathogen detection in a clinical environment, epidemiological investigation, or used to identify bio-terrorism agents.