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

The single-cell gel electrophoresis (Comet) assay is a relatively simple me thod of measuring DNA single strand breaks and alkali-labile sites in indiv idual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative inte grity on a single-cell basis. We show that the existence of strand disconti nuities in recently replicated domains of DNA, caused during semiconservati ve replication or exacerbated by the arrest of replicative polymerases at U V irradiation- or chemical-induced lesions, can be detected in individual c ells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves t he creation of strand breaks or gaps adjacent to recently replicated materi al, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al., The Le gacy of Cell Fusion, pp, 50-67, Oxford: Science Publications, 1994; R. B. P ainter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochem ical techniques, the BrdUrd-Comet assay is simple and suitable for the accu rate and automatable assessment of replicative integrity in very small numb ers of mammalian cells, such as may be obtained by biopsy.