Markus Durm

OPTIMIZATION OF FAST-FLUORESCENCE IN SITU HYBRIDIZATION WITH REPETITIVE ALPHA -SATELLITE PROBES

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.

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 α-Satellite Probe

Abstract 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 °C, hybridization was performed at 52 °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 °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 α-satellite DNA probes

Abstract 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 α-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°C hybridization temperature and 60 min hybridization time. For the Oncor chromosome-X-specific DNA probe, the optimal conditions were found at 74°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.

Painting of Human Chromosome 8 in Fifteen Minutes

Abstract The technique of chromosome-in-situ suppression (ClSS)-hybridization (chromosome painting) has now been well established. However, all standard protocols so far require long renaturation times (typically 12 hours and more). Here, we describe a new, extremely fast protocol for chromosome painting using a commercially available, directly fluorescence labelled probe for chromosome 8. The hybridization conditions used omit separate preannealing procedures and denaturing chemical agents. The renaturation time required for chromosome painting was reduced to 15 minutes. In addition, most washing steps were eliminated. As a consequence, the entire painting procedure was feasible in less than half an hour.

IN SITU ESTIMATES OF THE SPATIAL RESOLUTION FOR "PRACTICAL" FLUORESCENCE MICROSCOPY OF CELL NUCLEI

Axial and lateral responses obtained from ideal point objects through a fluorescence light microscope can be used to calculate the spatial resolution of the system from the point spread function. In practice, however, the experimental conditions given by a biological object can have an additional, considerable influence on the final resolution. Therefore, it is important to understand how parts of the setup contribute to the optimal response, e. g. cover glasses of different thickness, immersion and mounting media, or optical inhomogeneities of the biological, not point like objects themselves. Here, imaging properties of a confocal laser scanning fluorescence microscope are studied in situ in female lymphocyte cell nuclei. They were stained with a red fluorochrome (propidium iodide). Inside the nuclei, the centromeric regions of the two X chromosomes were specifically labelled by a green fluorochrome (FITC). Lateral and axial responses through the object and the labelling site were investigated. The increase or decrease (15%–85%) of the fluorescence intensities were used as an estimate for the spatial resolution of the system. This estimate was two times larger in the axial direction than in the lateral direction. The results suggest that such measurements can also be used in fluorescent biological objects as an internal standard to estimate the quality of resolution in “practical” quantitative fluorescence microscopy.