Multiple fluorescence in situ hybridization

Nederlof, Petra M.; Flier, S. van der; Wiegant, J.; Raap, Anton K.; Tanke, Hans J.; Ploem, J. S.; Ploeg, M. van der

doi:10.1002/cyto.990110115

Cited by 257 publications

(122 citation statements)

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“…The non-radioactive procedure has gained considerably in sensitivity during the last few years, and now allows detection of unique sequences of only a few kilobases (1,13, 20,22,32,34). The improved sensitivity, in combination with the development of methods for multiple ISH (i.e., the simultaneous detection of different sequences) (14, 23,25), has expanded the potential applications in genetics and molecular cell biology. Thus, whereas 10 years ago radioactive labels were used almost exclusively, non-radioactive labels such as fluorochromes and enzymes have now become more and more important for research and even clinical applications.…”

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Quantification of inter‐ and intra‐nuclear variation of fluorescence in situ hybridization signals

et al. 1992

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This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein.Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH.The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation.The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified. Furthermore, an internal standard to correct for differences in hybridization efficiency among cells, based on a second hybridization, was introduced.It is concluded that development of an accurate quantitative ISH procedure requires further reduction of the internuclear variation. Results presented indicate that improvements in the preparation of the target material and in the hybridization reaction are especially needed to fulfill this requirement. 0 1992 Wiley-Liss, Inc.

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Quantification of inter‐ and intra‐nuclear variation of fluorescence in situ hybridization signals

et al. 1992

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“…(1) In this protocol, all washing steps are performed in a Schifferdecker staining jar (commercially available) with a nominal volume of ~100 ml to cover slides. Other types of staining jar are also suitable and the amount of washing solutions should be re-calculated accordingly.…”

Section: Cobra-fishmentioning

confidence: 99%

“…Read and record absorption spectra, and use equation (1). ▲ CRITICAL STEP Typical DOL is between 1-3%; DOL below 1% will result in a weak signal, due to a low degree of dye incorporation, whereas DOL above 6-8% will result in a low signal due to quenching and loss of probes during posthybridization washing steps, caused by the decreased melting temperature of the labeled DNA.…”

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COBRA: combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization karyotyping

Szuhai

Tanke

2006

Nat Protoc

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Combined binary ratio labeling (COBRA) is designed to increase the multiplicity of fluorescence in situ hybridization (FISH)-i.e., the number of targets that can be distinguished simultaneously. In principle, chemical (ULS), enzymatic (nick translation or random priming) or PCR-based labeling procedures of probes can be used. The method was originally designed to label chromosome-painting probes, but has also been used for probe sets specific for subtelomeric regions.

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“…In addition, digital imaging devices, such as charge coupled device (CCD) cameras, contribute to increased sensitivity. Multiple haptenization and detection protocols, and combinatorial labeling approaches allow for the simultaneous visualization of several target regions in metaphase chromosomes and interphase nuclei (Nederlof et al 1990;Ried et al 1992a, b,c;Lenganer et al 1993;Wiegant et al 1993). The high spatial resolution of fluorescent signals is improved considerably when extended chromatin preparations are used for FISH analysis (Heng et al 1992;Wiegant et al 1992).…”

Section: Introductionmentioning

confidence: 99%

Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene

et al. 1994

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Abstract. We report on multicolor fluorescence in situ hybridization protocols for the simultaneous visualization of deletion-prone regions for carrier detection of Duchenne/ Becker (DMD/BMD) muscular dystrophy. Cosmid and yeast artificial chromosome (YAC) clones specific for preferentially deleted subregions of the dystrophin gene were labeled differentially and detected with three different fluorochromes using digital imaging microscopy. This approach allows for an assessment of the carrier status of female relatives even in families where no index patient is available. Cosmid and YAC clones, and different probegeneration protocols are compared with respect to their feasibility for cartier detection. The use of histone-depleted interphase nuclei (Halo-preparations) for deletion mapping is demonstrated and shown to have a resolution power of 5 kb.

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Multiple fluorescence in situ hybridization

Cited by 257 publications

References 16 publications

Quantification of inter‐ and intra‐nuclear variation of fluorescence in situ hybridization signals

Quantification of inter‐ and intra‐nuclear variation of fluorescence in situ hybridization signals

COBRA: combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization karyotyping

Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene

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