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Journal of Histochemistry and Cytochemistry, Vol. 48, 743-746, June 2000, Copyright © 2000, The Histochemical Society, Inc.

ARTICLE

DNA Fiber–FISH Staining Mechanism

Frans M. van de Rijkea, Ralph J. Florijnb, Hans J. Tankea, and Anton K. Raapa
a Departments of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands
b Human Genetics, Leiden University Medical Center, Leiden, the Netherlands

Correspondence to: Anton K. Raap, Lab. for Cytochemistry and Cytometry, Dept. of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. E-mail: a.k.raap@lumc.nl

Fluorescence in situ hybridization to DNA fibers (Fiber–FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber–FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotin-labeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber–FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes. (J Histochem Cytochem 48:743–745, 2000)

Key Words: DNA mapping, Fiber–FISH, hybridization


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