OPTIMIZED FAST-FISH WITH ALPHA-SATELLITE PROBES - ACCELERATION BY MICROWAVE ACTIVATION

It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization) technique is well suite d for quantitative microscopy. For highly repetitive DNA probes the hy bridization (renaturation) time and the number of subsequent washing s teps were reduced considerably by omitting denaturing chemical agents (e.g., formamide). The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical condit ions for hybridization permit automatization of the procedure, e.g., b y a programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific alpha-satellite probe. Highly fluorescent major binding sites were obtained for 74 degrees C hybrid ization temperature and 60 min hybridization time. They were clearly d iscriminated from some low fluorescent minor binding sites on metaphas e chromosomes as well as in interphase cell nuclei. On average, a tota l of 3.43 +/- 1.59 binding sites were measured in metaphase spreads, a nd 2.69 +/- 1.00 in interphase nuclei. Microwave activation for denatu ration and hybridization was tested to accelerate the procedure. The s lides with the target material and the hybridization buffer were place d in a standard microwave oven. After denaturation for 20 sec at 900 W , hybridization was performed for 4 min at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chr omosome 1-specific alpha-satellite probe. In this case, denaturation w as performed at 630 W for 60 sec and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clear ly discriminated by their brightness.