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The Comet Assay in Human Biomonitoring

  • Diana Anderson
  • Alok Dhawan
  • Julian Laubenthal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1044)

Abstract

Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects, and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single- and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate towards the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.

Key words

Comet assay Single-cell electrophoresis DNA damage Double-strand breaks Single-strand breaks Alkali-labile sites 

Notes

Acknowledgement

The funding from the UK India Education and Research Initiative (UKIERI) standard award to the University of Bradford, UK, and the Institute of Life Sciences, Ahmedabad University, India, is gratefully acknowledged.

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Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Diana Anderson
    • 1
  • Alok Dhawan
    • 2
    • 3
  • Julian Laubenthal
    • 4
  1. 1.Biomedical Sciences Division, School of Life SciencesUniversity of BradfordBradfordUK
  2. 2.Institute of Life Sciences, School of Science & TechnologyAhmedabad UniversityAhmedabadIndia
  3. 3.Nanomaterial Toxicology GroupCouncil of Scientific and Industrial Research (CSIR), Indian Institute of Toxicology ResearchLucknowIndia
  4. 4.Medical Sciences Division, School of Life SciencesUniversity of BradfordBradfordUK

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