OPTIMIZATION OF FAST-FLUORESCENCE IN-SITU HYBRIDIZATION WITH REPETITIVE ALPHA-SATELLITE PROBES

A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH ) for quantitative microscopy has been recently introduced. For highly repetitive DNA probes the hybridization (renaturation) time and the n umber of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low string ency conditions major and minor binding sites of the probes used showe d visible FISH signals well suited for quantitative image-microscopy. The discrimination of minor and major binding sites was possible by au tomated image-processing. Here, a further quantitative optimization of the Fast-FISH technique is described that allows to clearly discrimin ate major and minor binding sites of alpha-satellite probes by an easy image classification parameter. With respect to the optimization it w as necessary to verify two sensitive parameters (hybridization time an d temperature) of the given rapid FISH protocol. As examples the syste matic optimization for the two probes D 12 22 (major binding site on t he centromere of chromosome 12) and D 8 22 (major binding site on the centromere of chromosome 8) are shown. The optimal hybridization condi tions concerning rapidness and quality of chromosome morphology were o btained using a hybridization temperature of 70 degrees C and a hybrid ization time of 60 min. For these conditions major and minor binding s ites were clearly discriminated by the intensity maximum S-max of the corresponding FISH-spots.