The comet assay: topical issues

作者：Andrew Richard Collins, Á. Oscoz, Gunnar Brunborg, Isabel Gaivão, Lisa Giovannelli, Marcin K. Kruszewski, Catherine Choy Smith, Rudolf Štětina · 发表于：Mutagenesis · 年份：2008 · DOI：10.1093/mutage/gem051 · 被引用次数：990 · 研究领域：Carcinogens and Genotoxicity Assessment、RNA Interference and Gene Delivery、Molecular Biology Techniques and Applications

The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.