Mutagenesis Advance Access published online on April 2, 2009
Mutagenesis, doi:10.1093/mutage/gep009
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In vitro comet assay for DNA repair: a warning concerning application to cultured cells
Department of Nutrition, Faculty of Medicine, University of Oslo, Oslo, Norway
The comet assay (single-cell gel electrophoresis) is a sensitive and simple method for measuring DNA damage. An early modification of the assay involved the application of specific repair endonucleases to convert lesions to breaks; thus, for example, endonuclease III was used to measure oxidized pyrimidines. This concept has now been extended to produce an in vitro assay for DNA repair activity in a cell-free extract, for example from lymphocytes. The extract is incubated with substrate DNA containing specific base damage, and repair incision is detected as breaks in this DNA. We have recently been studying effects of phytochemicals in cultured cells, whether as antioxidants or as potential modulators of DNA repair. We realized that there is a need to check that observed effects that appear as an enhancement of repair (i.e. increased breaks in substrate DNA) are not simply due to a direct damaging effect of the phytochemical or to induction of non-specific nucleases. Here, we describe a rigorous approach to testing for this possibility, which we recommend to anyone carrying out similar experiments.
* To whom correspondence should be addressed. Department of Nutrition, University of Oslo, PB 1046 Blindern, 0316 Oslo, Norway. Tel: +47 22851329; Fax: +47 22851341; Email: o.a.azqueta{at}medisin.uio.no
Received on February 17, 2009; revised on March 4, 2009; accepted on March 4, 2009.