Mutagenesis vol. 19 no. 4 pp. 269-276,
July 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization
inská21Cancer Research Institute, Vlárska 7, 833 91 Bratislava, Slovakia, 2Institute of Preventive and Clinical Medicine, Slovak Medical University, Limbová 12, 833 03 Bratislava, Slovakia, 3Department of Nutrition, University of Oslo, PO Box 1046, Blindern, 0316 Oslo, Norway and 4Rowett Research Institute, Greenburn Road, Aberdeen AB21 9SB, UK
The comet assay is a sensitive method for measuring DNA strand breaks in eukaryotic cells. After embedding in agarose, cells are lysed and electrophoresed at high pH. DNA loops containing breaks (in which supercoiling is relaxed) escape from the nucleoid comet head to form a tail. Oligonucleotide probes were designed for 5' and 3' regions of the genes for dihydrofolate reductase (DHFR) and O6-methylguanine DNA methyltransferase (MGMT), both from the Chinese hamster, and the human tumour suppressor p53 gene. Alternate ends were labelled with either biotin or fluorescein. These probes were hybridized to the DNA of comets from Chinese hamster ovary (CHO) cells or human lymphocytes treated with H2O2 or photosensitizer plus light to induce oxidative damage. Amplification with Texas red- and fluorescein-tagged antibodies led, in the case of p53 in human cells, to red and green signals located in the comet tail (as well as in the head), indicating the presence of breaks in the vicinity of the gene. However, only one end of the MGMT gene appeared in the tail and almost no signals from the DHFR gene, either red or green, were in the tail of comets from CHO cells. Restriction on movement from the head to tail may result from the presence of a ‘matrix-associated region’ in the gene. The kinetics of repair of oxidative damage were followed; strand breaks in the p53 gene were repaired more rapidly than total DNA. Thus, fluorescent in situ hybridization in combination with the comet assay provides a powerful method for studying repair of specific genes in relation to chromatin structure.
5To whom correspondence should be addressed at: Department of Nutrition, University of Oslo, PO Box 1046, Blindern, 0316 Oslo, Norway. Tel: +47 22 85 13 60; Fax: +47 22 85 13 41; Email: a.r.collins{at}basalmed.uio.no
Received on March 7, 2003; revised on March 22, 2004; accepted on March 25, 2004
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