HUMAN CDNA MAPPING USING A HIGH-RESOLUTION R-BANDING TECHNIQUE AND FLUORESCENCE IN-SITU HYBRIDIZATION

High-resolution fluorescence in situ hybridization (FISH) is now an essential element in both human gene mapping and clinical cytogenetics. To facilitate its application, a series of techniques have been developed using FISH to map DNA probes in the size range of 1-1,000 kb directly on R-banded human chromosomes. Distinctive reverse (R) banding is achieved by staining with chromomycin A(3) and distamycin A following in situ hybridization. The use of such counterstains enables simultaneous viewing of both the fluorescent R-bands and in situ hybridization signals by either standard photomicroscopy or an automated image-acquisition system. This method is rapid and reproducibly reveals bands at the 350-700 stage. Further, specific methods for chromosome preparation, hybridization, and signal production have been developed and applied in combination with R-banding. These methods are used for precise chromosomal localization of DNA sequences in sizes ranging from that of cDNA (> 1 kb) through bacterial artificial chromosomes (100-150 kb) to yeast artificial chromosomes (greater than or equal to 1 Mb). These techniques provide high-resolution methods for rapid mapping of human genes, expanding the applications of FISH techniques in basic research and clinical analysis.