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Mitochondrial DNA as a Biosensor of UV Exposure in Human Skin

  • Amy Bowman
  • Mark A. Birch-Machin
Part of the Methods in Molecular Biology book series (MIMB, volume 1265)

Abstract

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage, including the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

Key words

Mitochondrial DNA Ultraviolet radiation Skin Real-time quantitative PCR Genetic damage 

Notes

Acknowledgment

This work was supported by the Institute of Cellular Medicine, the Faculty of Medical Sciences (Newcastle University), the North Eastern Skin Research Fund (NESRF), and the UK National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle Hospitals Foundation Trust and Newcastle University. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.

References

  1. 1.
    Kalinowski DP, Illenye S, Van Houten B (1992) Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. Nucleic Acids Res 20(13):3485–3494CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Ray AJ et al (2000) The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin. J Invest Dermatol 115(4):674–679CrossRefPubMedGoogle Scholar
  3. 3.
    Santos JH, Mandavilli BS, Van Houten B (2002) Measuring oxidative mtDNA damage and repair using quantitative PCR. Methods Mol Biol 197:159–176PubMedGoogle Scholar
  4. 4.
    Durham SE et al (2003) Mitochondrial DNA damage in non-melanoma skin cancer. Br J Cancer 88(1):90–95CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Eischeid AC, Meyer JN, Linden KG (2009) UV disinfection of adenoviruses: molecular indications of DNA damage efficiency. Appl Environ Microbiol 75(1):23–28CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Hunter SE et al (2010) The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number. Methods 51(4):444–451CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Swalwell H et al (2012) Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells. Free Radic Biol Med 52:626–634CrossRefPubMedGoogle Scholar
  8. 8.
    Birch-Machin MA, Swalwell H (2010) How mitochondria record the effects of UV exposure and oxidative stress using human skin as a model tissue. Mutagenesis 25(2):101–107CrossRefPubMedGoogle Scholar
  9. 9.
    Tulah AS, Birch-Machin MA (2013) Stressed out mitochondria: the role of mitochondria in ageing and cancer focussing on strategies and opportunities in human skin. Mitochondrion 13(5):444–453CrossRefPubMedGoogle Scholar
  10. 10.
    Oyewole AO et al (2014) Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells exposed to UVA and hydrogen peroxide. FASEB J 28(1):485–494CrossRefPubMedGoogle Scholar
  11. 11.
    Bowman A et al (2013) The simultaneous detection of mitochondrial DNA damage from sun-exposed skin of three whale species and its association with UV-induced microscopic lesions and apoptosis. Mitochondrion 13(4):342–349CrossRefPubMedGoogle Scholar
  12. 12.
    Martinez-Levasseur LM et al (2013) Whales use distinct strategies to counteract solar ultraviolet radiation. Sci Rep 3:2386CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Sikorsky JA et al (2007) DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency. Biochem Biophys Res Commun 355(2):431–437CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Koch H, Wittern KP, Bergemann J (2001) In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation. J Invest Dermatol 117(4):892–897CrossRefPubMedGoogle Scholar
  15. 15.
    Thermo Scientific UK (2013) 260/280 and 260/230 ratios. T009-Technical Bulletin NanoDrop 1000 & 8000. http://www.nanodrop.com/
  16. 16.
    Berdal KG, Holst-Jensen A (2001) Roundup Ready® soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses. Eur Food Res Technol 213(6):432–438Google Scholar
  17. 17.
    Niemitz EL et al (2004) Microdeletion of LIT1 in familial Beckwith-Wiedemann syndrome. Am J Hum Genet 75(5):844–849CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Mraz M et al (2009) MicroRNA isolation and stability in stored RNA samples. Biochem Biophys Res Commun 390(1):1–4CrossRefPubMedGoogle Scholar
  19. 19.
    Lee PY et al (2012) Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp (62). doi:10.3791/3923Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Dermatological Sciences, Institute of Cellular MedicineNewcastle UniversityNewcastle upon TyneUK

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