Single-strand breaks are among the most prevalent lesions found in DNA. Traditional electrophoretic methods (e.g., the Comet assay) used for investigating these lesions rely on alkaline conditions to denature DNA prior to electrophoresis. However, the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Herein, we describe a neutral glyoxal gel electrophoresis assay which is based on alkali-free DNA denaturation and is suitable for qualitative and semi-quantitative analyses of single-strand breaks in DNA isolated from different organisms.
Agarose Alkaline DNA damage DNA repair Electrophoresis Genotoxicant Glyoxal Neutral Single-strand breaks
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported in part by National Institute of Environmental Health Science grants (P42-ES05948, P30-ES10126).
Pitot HC, Dragan YP (1996) Chemical carcinogenesis. In: Klaassen CD (ed) Casarett and Doull’s toxicology, 5th edn. McGraw-Hill, New York, pp 201–267Google Scholar
Caldecott KW (2006) Mammalian single-strand break repair: mechanisms and links with chromatin. DNA Repair 6:443–453PubMedCrossRefGoogle Scholar
Georgiou C, Papapostolou I, Grizntzalis K (2009) Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks). Nat Protoc 4:125–131PubMedCrossRefGoogle Scholar
Luke AM et al (2010) Accumulation of true single strand breaks and AP sites in base excision repair deficient cells. Mutat Res 694:65–71PubMedCrossRefGoogle Scholar
McMaster GK, Carmichael GG (1977) Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A 74:4835–4838PubMedCrossRefGoogle Scholar
Drouin R, Gao S, Homquist GP (1996) Agarose gel electrophoresis for DNA damage analysis. In: Pfeifer GP (ed) Technologies for detection of DNA damage and mutations. Plenum Press, New York, pp 37–43CrossRefGoogle Scholar
Lin P-H et al (2003) Aldehydic DNA lesions induced by catechol estrogens in calf thymus DNA. Carcinogenesis 24:1133–1141PubMedCrossRefGoogle Scholar
Rodriguez H et al (2003) Mapping of peroxyl radical induced damage on genomic DNA. Biochemistry 38:16578–16588CrossRefGoogle Scholar
Pachkowski BF et al (2009) Cells deficient in PARP-1 show an accelerated accumulation of DNA single strand breaks, but not AP sites, over the PARP-1-proficient cells exposed to MMS. Mutat Res 671:93–99PubMedCrossRefGoogle Scholar
Nakamura J, La DK, Swenberg JA (2000) 5′-Nicked apurinic/apyrimidinic sites are resistant to beta-elimination by beta-polymerase and are persistent in human cultured cells after oxidative stress. J Biol Chem 275:5323–5328PubMedCrossRefGoogle Scholar
Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1:23–29PubMedCrossRefGoogle Scholar