Work proposed here will develop and apply an innovative approach to human gene mapping and the analysis of genetic disease using recently developed non-isotopic in situ hybridization methodology. This improved methodology makes possible the high-resolution fluorescence localization of single-copy genes or transcripts within interphase nuclei utilizing biotinated probes detected by fluorescein-avidin. This method provides significant advantages in resolution, speed and convenience for metaphase chromosome mapping as well, which will be demonstrated by precise metaphase localization of specific cellular genes, including cardiac myosin heavy chain and histone genes. Most importantly, however, the coupling of high resolution fluorescence microscopy with the decondensed nature of interphase chromatin makes it possible to resolve the linkage of DNA sequences separated by only 130 kb or less. """"""""Interphase Chromatin Mapping"""""""" has great potential to provide the rapid approximation of physical distances between cloned DNA sequences in a range encompassing several centimorgans (megabases) down to smaller distances of hundreds or tens of kb. The approach promises to be more cost-affective and less labor intensive than current techniques and may provide an accurate means of determining the closest flanking markers to a disease gene or of ordering tightly linked DNA sequences, as in construction of the one centimorgan genomic map. The limits of resolution of this technique will be defined and the relationship between interphase distance and DNA distance calibrated, using the well characterized Duchenne's Muscular Dystrophy locus as a model system. Interphase chromatin mapping will then be applied, as collaborative efforts, in an attempt to facilitate on-going searches for specific disease genes, particularly neurofibromatosis and familial polyposis coli. Secondary objectives of this proposal include analysis of higher- level nuclear organization and the further development of nuclear RNA detection techniques as a tool for investigating genetic disease or screening DNA sequences for expressed genes. Finally, the feasibility of applying high-quality, non-isotopic in situ hybridization to clinical diagnosis or cytogenetic research will be assessed.
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