A RAPID FISH TECHNIQUE FOR QUANTITATIVE MICROSCOPY

Results of quantitative microscopy for fluorescence in situ hybridizat ion (FISH) signals with repetitive DNA probes (pUC 1.77 and D15Z1) are reported. A nonenzymatic hybridization technique was applied using fl uorescein-12-dUTP labeled DNA probes and a buffer system not containin g any formamide or equivalent chemical denaturing agents. Following th ermal denaturation, the renaturation time was reduced to less than 30 min. The number of wash steps was reduced to one. For the pUC 1.77 pro be, the major binding sites (chromosome 1) were distinguished from the minor binding sites by means of fluorescence intensity and spot size. The intensity variation of the two brightest FISH spots (major bindin g sites) in the same metaphase was 19% for 15 min renaturation time an d 16% for 30 min renaturation time. For the D15Z1 probe, generally fou r bright spots were visible and tentatively assigned according to chro mosome length and centromere position (chromosomes 15 and 9). The inte nsity variation of each two homologues in the same metaphase spl ead s howed a coefficient of variation of 47% (15 min) and 22% (30 min) for chromosome 15, and 19% (15 min) and 15% (30 min) for chromosome 9. The results indicate that the applied technique can considerably accelera te the FISH procedure and is suited for quantitative microscopy.