*** 9661065 Trnovsky This Small Business Innovation Research Phase I project will explore development of a novel, ultrasensitive in situ hybridization format for detecting single copy genes in whole cells or nuclei encapsulated in agarose microdrops. A major goal of this research is to identify genes of interest present in a small fraction of the cell population without using target amplification. For single copy gene detection, the technology to be developed will compete primarily with methods based on target amplification, such as PCR. Although sensitive, these methods require release of target nucleic acids because purified templates are necessary for amplifications, making them unsuitable for cell-associated nucleic acid detection. This method to be researched will utilize novel chemiluminescent substrates for horseradish peroxidase in combination with a cooled CCD camera for detecting low light levels. Because no isolation of DNA is needed for in situ hybridizations, high signal densities are expected to permit detection of low DNA copy numbers at different cellular locations. The method can be used for identifying genes transferred to cellular genomes by in vitro (transfection, gene therapies) or in vivo (viral infections, transposition) methods and determining the percentage of cells containing transferred genes. Development of high sensitivity assay formats for detecting minute amounts of DNA for genomics research and molecular cytometics will contribute to development of economically important materials. ***
