An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity

Sona Vodenkova, Amaya Azqueta, Andrew Collins, Maria Dusinska, Isabel Gaivao, Peter Moller, Alena Opattova, Pavel Vodicka, Roger W. L. Godschalk, Sabine A. S. Langie*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

12 Citations (Web of Science)

Abstract

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.

This protocol describes a comet-based in vitro assay for detecting base and nucleotide excision repair activity for use in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues.

Original languageEnglish
Pages (from-to)3844-3878
Number of pages35
JournalNature Protocols
Volume15
Issue number12
Early online date16 Nov 2020
DOIs
Publication statusPublished - Dec 2020

Keywords

  • OXIDATIVELY DAMAGED DNA
  • CANCER PATIENTS
  • POLYPHENOLIC COMPOUNDS
  • OCCUPATIONAL-EXPOSURE
  • INCISION ACTIVITY
  • EXPRESSION LEVELS
  • TELOMERE LENGTH
  • MINERAL FIBERS
  • CROSS-LINKS
  • CELLS

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