RAPID FLUORESCENCE IN-SITU HYBRIDIZATION WITH REPETITIVE DNA PROBES -QUANTIFICATION BY DIGITAL IMAGE-ANALYSIS

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromos ome diagnostics. Here, results of a quantification of fluorescence sig nals after in situ hybridization with repetitive DNA probes are report ed using a non-enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA pro bes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH-signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphas e nuclei obtained from peripheral blood lymphocytes. The probes were l abelled with digoxigenin and detected by FITC-anti-digoxigenin. The hy bridization time was reduced step by step and the resulting fluorescen ce signals were examined systematically. For comparison the pUC 1.77 p robe was also hybridized according to a FISH protocol containing 50% f ormamide. By renaturation for 2 h and overnight two FISH signals per n ucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was per formed using a fluorescence microscope equipped with a cooled colour c harge coupled device (CCD) camera. Image analysis was made interactive ly using a commercially available software package running on a PC (80 486), For the pUC 1.77 probe the major binding sites (presumptive chro mosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the i nterphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation time s (0.5, 15, 30 min). The intra-nuclear variation in the intensity of t he two brightest in situ hybridization spots appeared to be slightly h igher (CV between 16 and 32%) than the corresponding variation on the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH si gnals were detected after hybridization without formamide and 15 min a nd 30 min renaturation. Always four bright spots were visible and tent atively assigned on the metaphase spreads (presumptive chromosome 15 a nd 9). The intensity variation of each pair of homologues in a metapha se spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromoso me 9. (C) 1994 Wiley-Liss, Inc.