FAST-FISH TECHNIQUE FOR RAPID, SIMULTANEOUS LABELING OF ALL HUMAN CENTROMERES

Fluorescence in situ hybridization (FISH) has become a powerful tool i n chromosome analysis. This report describes the systematic optimizati on of the Fast-FISH technique for centromere labelling of human metaph ase chromosomes for radiobiological dosimetry purposes, For the presen t study, the hybridization conditions and the efficiency of two commer cially available a-satellite DNA probes were compared (''human chromos ome 1 specific'', Oncor; Gaithersburg, MD, vs, ''all-human chromosomes specific'', Boehringer-Mannheim, Germany). These probes were hybridiz ed to human lymphocyte metaphase plates by using a hybridization buffe r without formamide and without any other equivalent denaturing chemic al agents, The results indicate the suitability of the method for auto mated image analysis on the basis of thresholding. The optimal conditi ons concerning hybridization time and temperature were determined by a systematic quantitative evaluation of the fluorescent labelling sites after the hybridization procedures, Under defined ''low stringency'' conditions, we found that the ''human chromosome 1 specific'' DNA prob e labelled not only the centromere of the human chromosome 1 but also the other human centromeres in the same way as the ''all-human chromos ome specific'' DNA probe, The optimized conditions to complete all cen tromere labelling were applied to the detection of dicentric chromosom es on irradiated human lymphocyte samples (gamma-rays of Co-60 source, 0.5 Gy/min, for doses of 1, 3, and 4 Gy), The yield of dicentrics was determined after Fast-FISH and compared with results obtained after G iemsa staining. These results are very compatible and indicate that, b ecause of its simplicity, this optimized Fast-FISH procedure would be useful for fast screening: purposes in biological dosimetry after acci dental overexposure. (C) 1998 Wiley-Liss, Inc.,Inc.