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Combining Fluorescent In Situ Hybridization with the Comet Assay for Targeted Examination of DNA Damage and Repair

  • Sergey Shaposhnikov
  • Preben D. Thomsen
  • Andrew R. CollinsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 682)

Abstract

The comet assay is a simple and sensitive method for measuring DNA damage. Cells are embedded in agarose on a microscope slide, lysed, and electrophoresed; the presence of strand breaks allows the DNA to migrate, giving the appearance of a comet tail, the percentage of DNA in the tail reflecting the break frequency. Lesion-specific endonucleases extend the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with damaging agent and monitoring the damage remaining at intervals during incubation. An important feature of the assay is that damage is detected at the level of individual cells. By combining the comet assay with fluorescent in situ hybridization (FISH), using labeled probes to particular DNA sequences, we can examine DNA damage and repair at the level of single genes or DNA sequences. Here we provide protocols for the comet assay and the FISH modification, answer some technical questions, and give examples of applications of the technique.

Key words

Comet assay Fluorescent in situ hybridization DNA damage DNA repair 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sergey Shaposhnikov
    • 1
  • Preben D. Thomsen
    • 2
  • Andrew R. Collins
    • 1
    Email author
  1. 1.Department of Nutrition, Faculty of MedicineUniversity of OsloOsloNorway
  2. 2.Faculty of Life ScienceUniversity of CopenhagenFrederiksbergDenmark

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