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An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity.
Vodenkova, Sona; Azqueta, Amaya; Collins, Andrew; Dusinska, Maria; Gaivão, Isabel; Møller, Peter; Opattova, Alena; Vodicka, Pavel; Godschalk, Roger W L; Langie, Sabine A S.
Afiliação
  • Vodenkova S; Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.
  • Azqueta A; Department of Medical Genetics, Third Faculty of Medicine, Charles University, Prague, Czech Republic.
  • Collins A; Department of Pharmacology and Toxicology, University of Navarra, and IdiSNA, Navarra Institute for Health Research, Pamplona, Spain.
  • Dusinska M; Department of Nutrition, University of Oslo, Oslo, Norway.
  • Gaivão I; Department of Environmental Chemistry, Health Effects Laboratory, NILU-Norwegian Institute for Air Research, Kjeller, Norway.
  • Møller P; Genetics and Biotechnology Department and Veterinary and Animal Research Centre (CECAV), Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal.
  • Opattova A; Section of Environmental Health, Department of Public Health, University of Copenhagen, Copenhagen, Denmark.
  • Vodicka P; Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.
  • Godschalk RWL; Biomedical Center, Medical Faculty in Pilsen, Charles University, Prague, Czech Republic.
  • Langie SAS; Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic.
Nat Protoc ; 15(12): 3844-3878, 2020 12.
Article em En | MEDLINE | ID: mdl-33199871
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.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Guideline Limite: Animals / Humans Idioma: En Ano de publicação: 2020 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Tipo de estudo: Guideline Limite: Animals / Humans Idioma: En Ano de publicação: 2020 Tipo de documento: Article