Measurement of DNA base and nucleotide excision repair activities in mammalian cells and tissues using the comet assay – A methodological overview |
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Authors: | Amaya Azqueta Sabine AS Langie Jana Slyskova Andrew R Collins |
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Institution: | 1. Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31009 Pamplona, Spain;2. Department of Nutrition, University of Oslo, PB 1046 Blindern, Oslo, Norway;3. Centre for Brain Ageing and Vitality, Institute for Ageing & Health, Human Nutrition Research Centre, Newcastle University, Campus for Ageing and Vitality, NE4 5PL Newcastle upon Tyne, UK;4. Department of Molecular Biology of Cancer, Institute of Experimental Medicine ASCR, Videnska 1083, Prague, Czech Republic |
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Abstract: | There is an increasing demand for phenotyping assays in the field of human functional genetics. DNA repair activity is representative of this functional approach, being seen as a valuable biomarker related to cancer risk. Repair activity is evaluated by incubating a cell extract with a DNA substrate containing lesions specific for the DNA repair pathway of interest. Enzymic incision at the lesion sites can be measured by means of the comet assay (single cell gel electrophoresis). The assay is particularly applicable for evaluation of base and nucleotide excision repair pathways (BER and NER). Substrate DNA containing oxidised purines gives a measure of BER, while UV-induced photolesions are the substrate for NER. While applications of comet-based DNA repair assays continue to increase, there are no commonly accepted standard protocols, which complicates inter-laboratory comparisons of results. |
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Keywords: | Comet assay Base excision repair Nucleotide excision repair Protocols Cell Tissue |
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