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Gene-Specific and Mitochondrial Repair of Oxidative DNA Damage

  • Protocol
DNA Repair Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 314))

Abstract

The Southern blot gene-specific DNA damage and repair assay is a robust and flexible method for quantifying many kinds of induced damage and repair with high reproducibility. Specific nicking and loss of a restricted DNA fragment at the site of induced damage is visualized by Southern blot and quantified against a control; since the blot is gene specific, only the damage of interest is measured. Here we show how the assay may be adapted to assess mitochondrial DNA (mtDNA) damage. In the mitochondrion, 8-oxoguanine is a significant oxidative lesion; in the laboratory, photoactivated methylene blue may be used to introduce this lesion into cells. Other lesions may also be studied by using different DNA damaging agents. We find that damage induction by methylene blue is consistently far greater in the mitochondrion than the nucleus. Thus advantageously, mitochondrial 8-oxoguanine repair may be studied without mtDNA isolation or preparation, which are processes known to induce DNA damage and skew measurements. This chapter gives detailed instructions for using methylene blue and the gene-specific repair assay to accurately measure mitochondrial oxidative damage and repair rates.

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Author information

Authors and Affiliations

  1. Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD

    R. Michael Anson, Penelope A. Mason & Vilhelm A. Bohr

Authors
  1. R. Michael Anson
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  2. Penelope A. Mason
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  3. Vilhelm A. Bohr
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Editors and Affiliations

  1. Pharmacological Sciences, SUNY Stony Brook, Stony Brook, NY

    Daryl S. Henderson

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© 2006 Humana Press Inc.

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Anson, R.M., Mason, P.A., Bohr, V.A. (2006). Gene-Specific and Mitochondrial Repair of Oxidative DNA Damage. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:155

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  • DOI: https://doi.org/10.1385/1-59259-973-7:155

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-513-2

  • Online ISBN: 978-1-59259-973-8

  • eBook Packages: Springer Protocols

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