Fast-FISH technique for rapid, simultaneous labeling of all human centromeres.

Fluorescence in situ hybridization (FISH) has become a powerful tool in chromosome analysis. This report describes the systematic optimization of the Fast-FISH technique for centromere labeling of human metaphase chromosomes for radiobiological dosimetry purposes. For the present study, the hybridization conditions and the efficiency of two commercially available alpha-satellite DNA probes were compared ("human chromosome 1 specific", Oncor, Gaithersburg, MD, vs. "all-human chromosomes specific", Boehringer-Mannheim, Germany). These probes were hybridized to human lymphocyte metaphase plates by using a hybridization buffer without formamide and without any other equivalent denaturing chemical agents. The results indicate the suitability of the method for automated image analysis on the basis of thresholding. The optimal conditions concerning hybridization time and temperature were determined by a systematic quantitative evaluation of the fluorescent labeling sites after the hybridization procedures. Under defined "low stringency" conditions, we found that the "human chromosome 1 specific" DNA probe labeled not only the centromere of the human chromosome 1 but also the other human centromeres in the same way as the "all-human chromosome specific" DNA probe. The optimized conditions to complete all centromere labeling were applied to the detection of dicentric chromosomes on irradiated human lymphocyte samples (gamma-rays of 60Co source, 0.5 Gy/min, for doses of 1, 3, and 4 Gy). The yield of dicentrics was determined after Fast-FISH and compared with results obtained after Giemsa staining. These results are very compatible and indicate that, because of its simplicity, this optimized Fast-FISH procedure would be useful for fast screening purposes in biological dosimetry after accidental overexposure.

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 suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation) time and the number of subsequent washing steps 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 conditions for hybridization permit automatization of the procedure, e.g., by 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 hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 +/- 1.59 binding sites were measured in metaphase spreads, and 2.69 +/- 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed 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 chromosome 1-specific alpha-satellite probe. In this case, denaturation was 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 clearly discriminated by their brightness.

Non-enzymatic, low temperature fluorescence in situ hybridization of human chromosomes with a repetitive alpha-satellite probe.

In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence, in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 degrees C, hybridization was performed at 52 degrees C for 30 min, i.e. at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 degrees C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF).

Optimization of Fast-FISH for alpha-satellite DNA probes.

It has been shown for several highly repetitive DNA probes that the newly introduced Fast-FISH (fast-fluorescence in situ hybridization) technique is well suited for quantitative microscopy. The advantage of omitting denaturing chemical agents (e.g., formamide) in the hybridization buffer results in a short hybridization time and a considerable reduction of the number of subsequent washing steps. Choosing the appropriate hybridization temperature and time allows to clearly discriminate major and minor binding sites by quantitative fluorescence microscopy. To further optimize the procedure with reference to reproducibility, a fully programmable thermal-cycler was applied for thermal de- and renaturation. Here, the optimized renaturation conditions for two commercially available alpha-satellite probes (specific for chromosomes 1 and X) are described. For the Boehringer chromosome-1-specific DNA probe, two highly fluorescent binding sites were obtained for 72 degrees C hybridization temperature and 60 min hybridization time. For the Oncor chromosome-X-specific DNA probe, the optimal conditions were found at 74 degrees C and 60 min hybridization time. In both cases the major binding sites were clearly discriminated from only a few weakly fluorescent minor binding sites on metaphase spreads as well as in interphase cell nuclei.

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 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 alpha-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. As examples the systematic optimization for the two probes D12Z2 (major binding site on the centromere of chromosome 12) and D8Z2 (major binding site on the centromere of chromosome 8) are shown. The optimal hybridization conditions concerning rapidness and quality of chromosome morphology were obtained using a hybridization temperature of 70 degrees C and a hybridization time of 60 min. For these conditions major and minor binding sites were clearly discriminated by the intensity maximum Smax of the corresponding FISH-spots.

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 chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported 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 probes, 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 interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC-anti-digoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 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 interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra-nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation in the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9.

A rapid FISH technique for quantitative microscopy.

Results of quantitative microscopy for fluorescence in situ hybridization (FISH) signals with repetitive DNA probes (pUC 1.77 and D15Z1) are reported. A nonenzymatic hybridization technique was applied using fluorescein-12-dUTP labeled DNA probes and a buffer system not containing any formamide or equivalent chemical denaturing agents. Following thermal 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 probe, 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 binding sites) in the same metaphase was 19% for 15 min renaturation time and 16% for 30 min renaturation time. For the D15Z1 probe, generally four bright spots were visible and tentatively assigned according to chromosome length and centromere position (chromosomes 15 and 9). The intensity variation of each two homologues in the same metaphase spread showed 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 accelerate the FISH procedure and is suited for quantitative microscopy.