Skip to main content
SpringerLink
Log in

Quantification of DNA Damage and Repair in Mitochondrial, Nuclear, and Bacterial Genomes by Real-Time PCR

  • Protocol
  • First Online:
Fast Detection of DNA Damage

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1644))

Abstract

DNA damage caused by genotoxic insults is often used as an indicator of specific diseases, environmental challenges, and metabolic processes. To date, various different methods have been described to detect damaged DNA. Many techniques need high amounts of DNA for the analysis and/or require the exact determination of DNA template concentration. Here, we describe a rapid and quantitative method for the evaluation of the relative levels of damage in mitochondrial, nuclear, and bacterial DNA in comparison to untreated controls. The approach is based on the real-time PCR amplification of DNA fragments of two different lengths in the respective samples. DNA damage detection using this protocol is gene-specific. The technique can also be expanded to monitor DNA repair and to detect genomic hot-spots for DNA lesions.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Niwa O (2003) Induced genomic instability in irradiated germ cells and in the offspring; reconciling discrepancies among the human and animal studies. Oncogene 22:7078–7086

    Article  CAS  PubMed  Google Scholar 

  2. Singh NP (2016) The comet assay: reflections on its development, evolution and applications. Mutat Res Rev 767:23–30

    Article  CAS  Google Scholar 

  3. Taghizadeh K, McFaline JL, Pang B et al (2008) Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nat Protoc 3:1287–1298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Santos JH, Meyer JN, Mandavilli BS et al (2006) Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol Biol 314:183–199

    Article  CAS  PubMed  Google Scholar 

  5. Rothfuss O, Gasser T, Patenge N (2010) Analysis of differential DNA damage in the mitochondrial genome employing a semi-long run real-time PCR approach. Nucleic Acids Res 38:e24

    Article  PubMed  Google Scholar 

  6. Sikorsky JA, Primerano DA, Fenger TW et al (2007) DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency. Biochem Biophys Res Commun 355:431–437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rothfuss O, Fischer H, Hasegawa T et al (2009) Parkin protects mitochondrial genome integrity and supports mitochondrial DNA repair. Hum Mol Genet 18:3832–3850

    Article  CAS  PubMed  Google Scholar 

  8. Artuso L, Zoccolella S, Favia P et al (2013) Mitochondrial genome aberrations in skeletal muscle of patients with motor neuron disease. Amyotroph Lateral Scler Frontotemporal Degener 14:261–266

    Article  CAS  PubMed  Google Scholar 

  9. Fendt L, Niederstatter H, Huber G et al (2011) Accumulation of mutations over the entire mitochondrial genome of breast cancer cells obtained by tissue microdissection. Breast Cancer Res Treat 128:327–336

    Article  CAS  PubMed  Google Scholar 

  10. Kowluru RA (2013) Mitochondria damage in the pathogenesis of diabetic retinopathy and in the metabolic memory associated with its continued progression. Curr Med Chem 20:3226–3233

    Article  CAS  PubMed  Google Scholar 

  11. Saydoff JA, Liu LS, Garcia RA et al (2003) Oral uridine pro-drug PN401 decreases neurodegeneration, behavioral impairment, weight loss and mortality in the 3-nitropropionic acid mitochondrial toxin model of Huntington's disease. Brain Res 994:44–54

    Article  CAS  PubMed  Google Scholar 

  12. Tewari S, Santos JM, Kowluru RA (2012) Damaged mitochondrial DNA replication system and the development of diabetic retinopathy. Antioxid Redox Signal 17:492–504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Carton-Garcia F, Riesco MF, Cabrita E et al (2013) Quantification of lesions in nuclear and mitochondrial genes of Sparus aurata cryopreserved sperm. Aquaculture 402-403:106–112

    Article  CAS  Google Scholar 

  14. Gonzalez-Rojo S, Fernandez-Diez C, Guerra SM et al (2014) Differential gene susceptibility to sperm DNA damage: analysis of developmental key genes in trout. PLoS One 9:e114161

    Article  PubMed  PubMed Central  Google Scholar 

  15. Valcarce DG, Carton-Garcia F, Riesco MF et al (2013) Analysis of DNA damage after human sperm cryopreservation in genes crucial for fertilization and early embryo development. Andrology 1:723–730

    Article  CAS  PubMed  Google Scholar 

  16. Edwards JG (2009) Quantification of mitochondrial DNA (mtDNA) damage and error rates by real-time QPCR. Mitochondrion 9:31–35

    Article  CAS  PubMed  Google Scholar 

  17. Lehle S, Hildebrand DG, Merz B et al (2013) LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis. Nucleic Acids Res 42:e41

    Article  PubMed  PubMed Central  Google Scholar 

  18. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  19. Navarro E, Serrano-Heras G, Castano MJ et al (2015) Real-time PCR detection chemistry. Clin Chim Acta 439:231–250

    Article  CAS  PubMed  Google Scholar 

  20. Furda A, Santos JH, Meyer JN et al (2014) Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol Biol 1105:419–437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingalle 70, Rostock, D-18057, Germany

    Nadja Patenge

Authors
  1. Nadja Patenge
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nadja Patenge .

Editor information

Editors and Affiliations

  1. Department of Neurosurgery and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA

    Vladimir V. Didenko

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Patenge, N. (2017). Quantification of DNA Damage and Repair in Mitochondrial, Nuclear, and Bacterial Genomes by Real-Time PCR. In: Didenko, V. (eds) Fast Detection of DNA Damage. Methods in Molecular Biology, vol 1644. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7187-9_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7187-9_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7185-5

  • Online ISBN: 978-1-4939-7187-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation