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Simultaneous Labeling of Single- and Double-Strand DNA Breaks by DNA Breakage Detection-FISH (DBD-FISH)

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DNA Damage Detection In Situ, Ex Vivo, and In Vivo

Part of the book series: Methods in Molecular Biology ((MIMB,volume 682))

Abstract

DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) permits simultaneous and selective labeling of single- and double-strand DNA breaks in individual cells, either in the whole genome or within specific DNA sequences. In this technique, cells are embedded into agarose microgels, lysed and subjected to electrophoresis under nondenaturing conditions. Subsequently, the produced “comets” are exposed to a controlled denaturation step which transforms DNA breaks into single-stranded DNA regions, detected by hybridization with whole genome fluorescent probes or the probes to specific DNA sequences. This makes possible a targeted analysis of various chromatin areas for the presence of DNA breaks. The migration length of the DBD-FISH signal is proportional to the number of double strand breaks, whereas its fluorescence intensity depends on numbers of single-strand breaks.

The detailed protocol for detection of two types of DNA breaks produced by ionizing radiation is presented. The technique can be used to determine intragenomic and intercellular heterogeneity in the induction and repair of DNA damage.

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Acknowledgments

This work was supported by Fondo de Investigaciones Sanitarias (FIS PI070459), Xunta de Galicia (INCITE07PXI916201ES) and the Consejo de Seguridad Nuclear (CSN), Spain.

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Authors and Affiliations

  1. INIBIC-Complexo Hospitalario Universitario A Coruña (CHUAC), Centro Oncológico de Galicia, La Coruña, Spain

    José Luis Fernández

  2. INIBIC-Complexo Hospitalario Universitario A Coruña (CHUAC), La Coruña, Spain

    Dioleyda Cajigal

  3. Facultad de Biología, Universidad Autónoma de Madrid, Madrid, Spain

    Jaime Gosálvez

Authors
  1. José Luis Fernández
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  2. Dioleyda Cajigal
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  3. Jaime Gosálvez
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Editor information

Editors and Affiliations

  1. Baylor College of Medicine, Depts. Neurosurgery and Molecular & Cell, University of Houston, Holcombe Blvd. 2002, Houston, 77030, Texas, USA

    Vladimir V. Didenko

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Fernández, J.L., Cajigal, D., Gosálvez, J. (2011). Simultaneous Labeling of Single- and Double-Strand DNA Breaks by DNA Breakage Detection-FISH (DBD-FISH). In: Didenko, V. (eds) DNA Damage Detection In Situ, Ex Vivo, and In Vivo. Methods in Molecular Biology, vol 682. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-409-8_11

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  • DOI: https://doi.org/10.1007/978-1-60327-409-8_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-408-1

  • Online ISBN: 978-1-60327-409-8

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