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Fluorescence In Situ Hybridization on Electrophoresed Cells to Detect Sequence Specific DNA Damage

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DNA Electrophoresis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1054))

Abstract

Fluorescence in situ hybridization (FISH) to label fragments of DNA with probes which can specifically locate a genomic region of interest, combined with the single cell electrophoresis (Comet) assay, also termed Comet-FISH, allows the quantification of DNA damage and repair at a specific genomic locus. While the Comet assay alone quantifies only the overall DNA damage of an individual cell, subsequent FISH on the electrophoresed single cell genome enables the coincidental localization of fluorescently labelled sequences (i.e., probes) to the respective damaged or undamaged genes or specific genomic regions of interest. In that way sequence specific DNA damage, global genomic and transcription coupled repair or the three dimensional ultrastructure of cells from any tissue can be comparatively investigated. This protocol provides a detailed description of the principles and basic methodology of a standard Comet-FISH experiment to study interphase cells of any tissue. Also important variations of the protocol (e.g., neutral conditions to detect double strand breaks) as well as the production of fluorochrome-labelled DNA probes via random priming are described.

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Authors and Affiliations

  1. School of Life Sciences, University of Bradford, Bradford, UK

    Julian Laubenthal & Diana Anderson

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  1. Julian Laubenthal
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  2. Diana Anderson
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Editors and Affiliations

  1. Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom

    Svetlana Makovets

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Laubenthal, J., Anderson, D. (2013). Fluorescence In Situ Hybridization on Electrophoresed Cells to Detect Sequence Specific DNA Damage. In: Makovets, S. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 1054. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-565-1_15

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  • DOI: https://doi.org/10.1007/978-1-62703-565-1_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-564-4

  • Online ISBN: 978-1-62703-565-1

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