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DNA Repair Protocols pp 111–132Cite as

Analysis of DNA Damage and Repair in Nuclear and Mitochondrial DNA of Animal Cells Using Quantitative PCR

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 920))

Abstract

This chapter was written as a guide to using the long-amplicon quantitative PCR (QPCR) assay for the measurement of DNA damage in mammalian as well as nonmammalian species such as Caenorhabditis elegans (nematodes), Drosophila melanogaster (fruit flies), and two species of fish (Fundulus heteroclitus and Danio rerio). Since its development in the early 1990s (Kalinowski et al., Nucleic Acids Res 20:3485–3494, 1992; Salazar and Van Houten, Mutat Res 385:139–149, 1997; Yakes and Van Houten, Proc Natl Acad Sci USA 94:514–519, 1997), the QPCR assay has been widely used to measure DNA damage and repair kinetics in nuclear and mitochondrial genomes after genotoxin exposure (Yakes and Van Houten, Proc Natl Acad Sci USA 94:514–519, 1997; Santos et al., J Biol Chem 278:1728–1734, 2003; Mandavilli et al., Mol Brain Res 133:215–223, 2005). One of the main strengths of the assay is that the labor-intensive and artifact-generating step of mitochondrial isolation is not needed for the accurate measurement of mitochondrial DNA copy number and damage. Below we present the advantages and limitations of using QPCR to assay DNA damage in animal cells and provide a detailed protocol of the QPCR assay that integrates its usage in newly developed animal systems.

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Author information

Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Amy M. Furda

  2. Nicholas School of the Environment, Duke University, Durham, NC, USA

    Amanda Smith Bess & Joel N. Meyer

  3. Department of Pharmacology and Chemical Biology, Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, USA

    Bennett Van Houten

Authors
  1. Amy M. Furda
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  2. Amanda Smith Bess
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  3. Joel N. Meyer
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  4. Bennett Van Houten
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Corresponding author

Correspondence to Bennett Van Houten .

Editor information

Editors and Affiliations

  1. , Department of Molecular Biology, Aarhus University, C.F Møllers Allé bygning 1130, Århus C, 8000, Denmark

    Lotte Bjergbæk

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Furda, A.M., Bess, A.S., Meyer, J.N., Van Houten, B. (2012). Analysis of DNA Damage and Repair in Nuclear and Mitochondrial DNA of Animal Cells Using Quantitative PCR. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_9

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  • DOI: https://doi.org/10.1007/978-1-61779-998-3_9

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-997-6

  • Online ISBN: 978-1-61779-998-3

  • eBook Packages: Springer Protocols

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