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    Analysis of DNA damage via single-cell electrophoresis

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    Publication date
    2013
    Author
    Anderson, Diana
    Laubenthal, Julian
    Keyword
    Comet assay; Single-cell electrophoresis; DNA damage; Double-strand breaks; Single-strand breaks; Alkali-labile sites
    Peer-Reviewed
    Yes
    
    Metadata
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    Abstract
    The comet assay or single-cell gel electrophoresis assay is a relatively simple and sensitive technique for quantitatively measuring DNA damage and repair at the single-cell level in all types of tissue where a single-cell suspension can be obtained. Isolated cells are mixed with agarose, positioned on a glass slide, and then lysed in a high-salt solution which removes all cell contents except the nuclear matrix and DNA, which is finally subjected to electrophoresis. Damaged DNA is electrophoresed from the nuclear matrix into the agarose gel, resembling the appearance of a comet, while undamaged DNA remains largely within the proximity of the nuclear matrix. By choosing different pH conditions for electrophoresis, different damage types and levels of sensitivity are produced: a neutral (pH 8–9) electrophoresis mainly detects DNA double-strand breaks, while alkaline (pH ≥ 13) conditions detect double- and single-strand breaks as well as alkali-labile sites. This protocol describes a standard comet assay study for the analysis of DNA damage and outlines important variations of this protocol.
    URI
    http://hdl.handle.net/10454/10115
    Version
    No full-text available in the repository
    Citation
    Anderson D and Laubenthal J (2013) Analysis of DNA damage via single-cell electrophoresis. In: Makovets S (Ed) DNA Electrophoresis. (Methods in Molecular Biology) New York: Humana Press: 209-218.
    Link to publisher’s version
    http://dx.doi.org/10.1007/978-1-62703-565-1_14
    Type
    Book chapter
    Collections
    Life Sciences Publications

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