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During the last decade, genome sequence databases of many species have been more and more completed so that it has become possible to further develop a recently established technique of FISH (Fluorescence In Situ Hybridization) called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to standard FISH techniques, COMBO-FISH makes use of a bioinformatic search in sequence databases for probe design, so that it can be done for any species so far sequenced. In the original approach, oligonucleotide stretches of typical lengths of 15-30 nucleotides were selected in such a way that they only co-localize at the given genome target. Typical probe sets of about 20-40 stretches were used to label about 50-250 kb specifically. The probes of different lengths can be composed of purines and pyrimidines, but were often restricted to homo-purine or homo-pyrimidine probe sets because of the experimental advantage of using a protocol omitting denaturation of the target strand and triple strand binding of the probes. This allows for a better conservation of the 3D folding and arrangement of the genome. With an improved, rigorous genome sequence database analysis and sequence search according to statistical frequency and uniqueness, a novel family of probes repetitively binding to characteristic genome features like SINEs (Short Interspersed Nuclear Elements, e.g., ALU elements), LINEs (Long Interspersed Nuclear Elements, e.g., L1), or centromeres has been developed. These probes can be synthesized commercially as DNA or PNA probes with high purity and labeled by fluorescent dye molecules. Here, new protocols are described for purine-pyrimidine probes omitting heat treatment for denaturation of the target so that oligonucleotide labeling can also be combined with immune-staining by specific antibodies. If the dyes linked to the oligonucleotide stretches undergo reversible photo-bleaching (laser-induced slow blinking), the labeled cell nuclei can be further subjected to super-resolution localization microscopy for complex chromatin architecture research.

Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements-dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA-DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets.

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy., (Copyright © 2016. Published by Elsevier Inc.)

Trinucleotide repeat expansions (like (CGG)n) of chromatin in the genome of cell nuclei can cause neurological disorders such as for example the Fragile-X syndrome. Until now the mechanisms are not clearly understood as to how these expansions develop during cell proliferation. Therefore in situ investigations of chromatin structures on the nanoscale are required to better understand supra-molecular mechanisms on the single cell level. By super-resolution localization microscopy (Spectral Position Determination Microscopy; SPDM) in combination with nano-probing using COMBO-FISH (COMBinatorial Oligonucleotide FISH), novel insights into the nano-architecture of the genome will become possible. The native spatial structure of trinucleotide repeat expansion genome regions was analysed and optical sequencing of repetitive units was performed within 3D-conserved nuclei using SPDM after COMBO-FISH. We analysed a (CGG)n-expansion region inside the 5' untranslated region of the FMR1 gene. The number of CGG repeats for a full mutation causing the Fragile-X syndrome was found and also verified by Southern blot. The FMR1 promotor region was similarly condensed like a centromeric region whereas the arrangement of the probes labelling the expansion region seemed to indicate a loop-like nano-structure. These results for the first time demonstrate that in situ chromatin structure measurements on the nanoscale are feasible. Due to further methodological progress it will become possible to estimate the state of trinucleotide repeat mutations in detail and to determine the associated chromatin strand structural changes on the single cell level. In general, the application of the described approach to any genome region will lead to new insights into genome nano-architecture and open new avenues for understanding mechanisms and their relevance in the development of heredity diseases.

With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.

With the improvement and completeness of genome databases, it has become possible to develop a novel fluorescence in situ hybridization (FISH) technique called COMBinatorial Oligo FISH (COMBO-FISH). In contrast to other (standard) FISH applications, COMBO-FISH makes use of a bioinformatic approach for probe set design. By means of computer genome database search, oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that they all colocalize within a given genome (gene) target. Typically, probe sets of about 20-40 stretches are designed within 50-250 kb, which is enough to get an increased fluorescence signal specifically highlighting the target from the background. Although "specific colocalization" is the only necessary condition for probe selection, i.e. the probes of different lengths can be composed of purines and pyrimidines, we additionally refined the design strategy restricting the probe sets to homopurine or homopyrimidine oligonucleotides so that depending on the probe orientation either double (requiring denaturation of the target double strand) or triple (omitting denaturation of the target strand) strand bonding of the probes is possible. The probes used for the protocols described below are DNA or PNA oligonucleotides, which can be synthesized by established automatized techniques. We describe different protocols that were successfully applied to label gene targets via double- or triple-strand bonding in fixed lymphocyte cell cultures, bone marrow smears, and formalin-fixed, paraffin-wax embedded tissue sections. In addition, we present a procedure of probe microinjection in living cells resulting in specific labeling when microscopically detected after fixation.

Purpose: HER2 may be a relevant biomarker in Barrett's cancer. We compared three HER2 laboratory methods, standard fluorescence in situ hybridization (FISH), image-based three-dimensional FISH in thick (16 microm) sections, and immunohistochemistry, to predict patient outcome., Experimental Design: Tissue microarray sections from 124 Barrett's cancer patients were analyzed by standard FISH on thin (4 microm) sections and by image-based three-dimensional FISH on thick (16 microm) sections for HER2 and chromosome-17, as well for p185(HER2) by immunohistochemistry. Correlations with clinical and follow-up data were examined., Results: Only three-dimensional FISH on thick (16 microm) sections revealed HER2 gene copy gain to be associated with increased disease-specific mortality (relative risk, 2.1; 95% confidence interval, 1.06-4.26; P = 0.033). In contrast, standard FISH on thin (4 microm) sections and immunohistochemistry failed to predict clinical outcome. Low-level gain of HER2 occurred frequently in Barrett's cancer (>or=2.5-4.0 HER2 copies, 59.7%; HER2-to-chromosome-17 ratio, >or=1.1-2.0; 61.2%) and defined a subpopulation for patient outcome as unfavorable as HER2 gene amplification [disease-free survival, P = 0.017 (HER2 copies)]. This low-level group was neither definable by standard FISH nor immunohistochemistry. No prognostic significance was found for chromosome-17 aneusomy., Conclusions: Low-level copy gains of HER2 define a biologically distinct subpopulation of Barrett's cancer patients. Importantly, these subtle copy number changes are not reliably detected by standard FISH in thin (4 microm) tissue sections, highlighting a thus far unrecognized weakness in HER2 FISH testing. These results should be taken into account for accurate evaluation of biomarkers by FISH and for HER2 FISH testing in tissue sections.

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an approach.

Structural analysis and nanosizing of gene domains requires not only high-resolution microscopy but also improved techniques of fluorescence labelling strongly focussed on the gene domains. To investigate the architecture of abl and bcr in blood cell nuclei forming the Philadelphia chromosome in CML, we applied COMBO-FISH using specifically colocalising combinations of triple strand forming oligonucleotide probes for abl on chromosome 9 and bcr on chromosome 22. Each probe set consisting of 31 homopyrimidine oligonucleotides was computer selected from the human genome database. Measurements by 3D microscopy were compared to results obtained after standard FISH using commercially available abl/bcr BAC probes. The relative radial fluorescence distributions in lymphocyte cell nuclei of healthy donors in comparison to cell nuclei of blood cells of CML patients showed a strong correlation in the location of abl and bcr for both labelling techniques. The absolute distances of the homologous bcr domains and the abl domain-nuclear center-abl domain angles in cell nuclei of CML donors differed significantly from those of healthy donors only when COMBO-FISH was applied. These results indicate that COMBO-FISH may be more sensitive than standard FISH in case of slight modifications in the genome architecture.

The comet-FISH technique described in this protocol is a tool to detect genome region-specific DNA damage and repair. It is a combination of two established techniques, the comet assay (or single-cell gel electrophoresis, or the single-cell gel test), to separate highly fragmented from moderately or nonfragmented DNA and to measure it, and fluorescence in situ hybridization (FISH), to specifically label DNA sequences of interest. Comet-FISH exists in two versions, based on the neutral and the alkaline comet assays. A detailed description of the comet assay is given in Chapter 9, so readers who are not familiar with this technique can work directly with the protocol described here, without referring to additional protocols reported elsewhere. The neutral version of the comet assay detects double-strand breaks, while the alkaline version detects both double- and single-strand breaks as well as abasic sites or sites of incomplete repair. This chapter also details cell preparation and production of the hybridization probes adapted to the comet-FISH technique. Finally, microscopic analysis of comet-FISH results is described, and possible procedures of quantification of the specific DNA damage are presented.

Here we present the principle of fluorescence in situ hybridization (FISH) with combinatorial oligonucleotide (COMBO) probes as a new approach for the specific labeling of genomic sites. COMBO-FISH takes advantage of homopurine/homopyrimidine oligonucleotides that form triple helices with intact duplex genomic DNA, without the need for prior denaturation of the target sequence that is usually applied for probe binding in standard FISH protocols. An analysis of human genome databases has shown that homopurine/homopyrimidine sequences longer than 14 bp are nearly homogeneously distributed over the genome, and they represent from 1% to 2% of the entire genome. Because the observation volume in a confocal laser-scanning microscope equipped with a high numerical aperture lens typically corresponds to an approximate 250-kb chromatin domain in a normal mammalian cell nucleus, this volume should contain 150-200 homopurine/homopyrimidine stretches. Using DNA database information, one can configure a set of distinct, uniformly labeled oligonucleotide probes from these stretches that is expected to exclusively co-localize within a 250-kb chromatin domain. Due to the diffraction-limited resolution of a microscope, the fluorescence signals of the configured oligonucleotide probe set merge into a typical, nearly homogenous FISH spot. Using a set of 32 homopyrimidine probes, we performed experiments in the Abelson murine leukemia region of human chromosome 9 as some of the very first proofs-of-principle of COMBO-FISH. Although the experimental protocol currently contains several steps that are incompatible with living cell conditions, the theoretical approach may be the first methodological advance toward the long-term but still elusive goal of carrying out specific FISH in high-resolution fluorescence microscopy of vital cells.

A non-enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context 'low temperature' means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 degrees C. For LTFISH, the DNA probe had to be single-stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near-field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH-procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single-stranded DNA probes to double-stranded DNA targets are discussed.

Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.

For chromosome painting, in situ suppression of repetitive DNA sequences has been well established. Such standard protocols usually require large amounts of Cot-I DNA. Recently, it has become possible to deplete repetitive DNA sequences from library probes by magnetic purification and PCR-assisted affinity chromatography. These "repeat-depleted library probes" appear to be extremely useful for Fast-FISH, a technique that omits denaturing chemical agents such as formamide in the hybridization buffer, resulting in a substantial acceleration and simplification of the complete protocol. Shown here is the application of Fast-FISH to a repeat-depleted, directly fluorochrome-labeled library probe of the q-arm of chromosome 15 (Fast-Painting) for human lymphocyte metaphase spreads. Following painting without Cot-I DNA and without formamide, visual inspection revealed sufficient chromosome painting after a few hours of hybridization. The fluorescence signals of the labeling sites were analyzed after hybridization times of 1 and 2 h (in one case, 4 h) using digital fluorescence microscopy. The painting efficiency expressed in values of relative fluorescence signal ratios was quantitatively evaluated by image analysis using line-scan procedures and area-morphometry of mean luminance. Two preparation protocols (ethanol dehydration without and with RNase A treatment followed by pepsin digestion for four different exposure times) were compared. These results indicated that RNase A treatment and pepsin digestion are steps that can be omitted.

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.

A new fluorescence in situ hybridization (FISH) technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL). Commercially available alpha-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, and microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

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.

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.

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.

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.

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.