Aging Aging

Comet Assay of DNA Damage 14310Measurement of DNA Damage and Repair Capacityas a Function of Age Using the Comet AssayPeter H. Clingen, Jillian E. Lowe, and Michael H. L. Green1. IntroductionA large number of studies indicate that DNA damage and mutation increasewith age in human cells and tissues (1). Age-related degenerative disorders inwhich DNA damage has been invoked include heart disease andneurodegenerative conditions such as Alzheimer’s disease, amyotrophic lateralsclerosis, or Parkinson’s disease (2,3). Patients with deficiencies in DNA repair,including xeroderma pigmentosum (XP) (4) and ataxia–telangiectasia (A–T)(5) show characteristic patterns of neurodegeneration (as opposed to a failureof normal development). The implication is that failure of repair can lead toaccumulation of damage and degenerative disease. XPs and A–Ts are hypersensitiveto specific types of DNA damage, and the degenerative damage inpatients is tissue specific. DNA in every tissue, however, is under attack from arange of endogenously formed mutagens, including reactive oxygen species,nitric oxide, reactive metabolites, and breakdown products such as malondialdehyde.A series of repair enzymes recognize and remove these types ofDNA damage from the genome. Failure to repair DNA may cause the synthesisof defective proteins, which will repair DNA less efficiently, and in turn lead topropagation of further errors (6). Alternatively, oxidative damage to mitochondrialproteins might cause less efficient processing of oxygen, release of higherlevels of reactive oxygen species and increased levels of background DNAdamage.In this chapter, we describe protocols based on modifications of the alkaliComet assay (single-cell gel electrophoresis) and describe how they may beapplied to the measurement of DNA damage and repair as a function of age.The Comet assay is a simple, rapid, and inexpensive method for detecting DNAFrom: Methods in Molecular Medicine, Vol. 38: Aging Methods and ProtocolsEdited by: Y. A. Barnett and C. R. Barnett © Humana Press Inc., Totowa, NJ143