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The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells

Biomedical Sciences Research Institute Computer Science Research Institute Environmental Sciences Research Institute Nanotechnology & Advanced Materials Research Institute

McGlynn, AP, Wasson, G, O'Connor, Jacqueline, McKerr, George, McKelvey-Martin, Valerie and Downes, Stephen (1999) The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells. CANCER RESEARCH, 59 (23). pp. 5912-5916. [Journal article]

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Abstract

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al., The Legacy of Cell Fusion, pp, 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.

Item Type:Journal article
Faculties and Schools:Faculty of Life and Health Sciences
Faculty of Life and Health Sciences > School of Biomedical Sciences
Faculty of Life and Health Sciences > School of Pharmacy and Pharmaceutical Science
Research Institutes and Groups:Biomedical Sciences Research Institute
Biomedical Sciences Research Institute > Molecular Medicine
Biomedical Sciences Research Institute > Molecular Medicine > Nano Systems Biology
ID Code:3402
Deposited By:Professor Stephen Downes
Deposited On:15 Dec 2009 13:58
Last Modified:04 Dec 2012 12:28

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