Optimization of Fast-Fluorescence in situ Hybridization with Repetitive α-Satellite Probes

Abstract

Abstract A rapid FISH (fluorescence in situ hybridization) technique (Fast-FISH) for quantitative microscopy has been recently introduced. For highly repetitive D N A probes the hybridization (renaturation) time and the number of necessary washing steps were reduced considerably by omitting formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes used showed visible FISH signals well suited for quantitative image-microscopy. The discrimination of minor and major binding sites was possible by automated image-processing. Here, a further, quantitative optimization of the Fast-FISH technique is described that allows to clearly discriminate major and minor binding sites of a-satellite probes by an easy image classification parameter. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature) of the given rapid FISH protocol. A s examples the systematic optimization for the two probes D 12 Z 2 (major binding site on the centromere of chromosome 12) and D 8 Z 2 (major binding site on the centromere of chromosom e 8) are shown. The optimal hybridiza­tion conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 °C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maxi­ mum Smax of the corresponding FISH-spots.