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

AP McGlynn, G Wasson, Jacqueline O'Connor, George McKerr, Valerie McKelvey-Martin, Stephen Downes

Research output: Contribution to journalArticle

29 Citations (Scopus)

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.
LanguageEnglish
Pages5912-5916
JournalCancer Research
Volume59
Issue number23
Publication statusPublished - Dec 1999

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Comet Assay
Bromodeoxyuridine
DNA
Single-Stranded DNA Breaks
Cell Fusion
Alkalies
DNA-Directed DNA Polymerase
DNA Replication
DNA Damage
Publications
Cell Count
Biopsy

Cite this

McGlynn, AP., Wasson, G., O'Connor, J., McKerr, G., McKelvey-Martin, V., & Downes, S. (1999). The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells. Cancer Research, 59(23), 5912-5916.
McGlynn, AP ; Wasson, G ; O'Connor, Jacqueline ; McKerr, George ; McKelvey-Martin, Valerie ; Downes, Stephen. / The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells. In: Cancer Research. 1999 ; Vol. 59, No. 23. pp. 5912-5916.
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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.",
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McGlynn, AP, Wasson, G, O'Connor, J, McKerr, G, McKelvey-Martin, V & Downes, S 1999, 'The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells', Cancer Research, vol. 59, no. 23, pp. 5912-5916.

The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells. / McGlynn, AP; Wasson, G; O'Connor, Jacqueline; McKerr, George; McKelvey-Martin, Valerie; Downes, Stephen.

In: Cancer Research, Vol. 59, No. 23, 12.1999, p. 5912-5916.

Research output: Contribution to journalArticle

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McGlynn AP, Wasson G, O'Connor J, McKerr G, McKelvey-Martin V, Downes S. The bromodeoxyuridine comet assay: Detection of maturation of recently replicated DNA in individual cells. Cancer Research. 1999 Dec;59(23):5912-5916.