University of California Water Resources Center

The advent of molecular-based diagnostics has radically improved our ability to analyze the prevalence and activities of microorganisms in environmental systems. In this research. a new molecular tool (gene probe spectroscopy or GPS) was tested which may further enhance the capabilities of existing molecular techniques for carrying-out environmental analysis. With GPS, a microorganism (or a group or related microorganisms) is characterized by its hybridization to a set of non-specific oligonucleotide probes in a dot-blot format. The hybridization intensities for each probe/microorganism combination is determined experimentally and stored in a matrix database (A). Samples containing unknown mixtures of microorganisms are then hybridized with the same set of probes to obtain a set of composite hybridization intensities (b). By employing linear inverse theory, the concentration (c) of each microorganism present in the unknown sample may be computed by matrix inversion techniques: C = A -lb. With an award from the Water Resources Center, we investigated this approach using a model system consisting of four different bacteriophage (T2, M13, lambda, and 1'7) and six non-specific oligonucleotide probes each of which cross-reacts with two or more phage. In a series of tests, phage concentrations were correctly predicted by the method. illustrating its efficacy for identifying and enumerating microorganisms in fluid samples. Its application to "real" environments will require the use of DNA chip technology in which oligonucleotide probes are arrayed on glass slides. Future work will be directed toward the merging of the techniques developed in this project, namely GPS, with the DNA chip format,