Department of General and Molecular Genetics, National Shevchenko University, Kiev, Ukraine.
Electrophoresis. 2010 Jan;31(3):512-9. doi: 10.1002/elps.200900421.
We investigated the mechanisms of DNA exit during single-cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single-strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.
我们通过测量彗星尾部形成的动力学来研究单细胞凝胶电泳（彗星试验）中 DNA 外溢的机制。在中性彗星试验中，发现 DNA 外溢的速度取决于 DNA 的拓扑状态，而拓扑状态受溴化乙锭或低辐射剂量的影响。结果清楚地表明，彗星尾部是由伸展的 DNA 环形成的：当 DNA 扭转约束保持在环中时，环延伸是可逆的，当约束松弛时，环延伸是有利的。在高辐射剂量的情况下，彗星形成的动力学表明，单链断裂的积累导致 DNA 片段化。与中性彗星试验不同，碱性彗星试验与染色质环无关。我们的结果表明，碱性处理会导致环从核基质上脱离，彗星尾部由 ssDNA 片段形成，其末端通过电力从彗星头部被拉出。我们建议，动力学方法可以被视为彗星试验的一个重要改进。
