Kinetics of comet formation in single-cell gel electrophoresis: loops and fragments.
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.
Afanasieva
Katerina
K
Department of General and Molecular Genetics, National Shevchenko University, Kiev, Ukraine.
Zazhytska
Marianna
M
Sivolob
Andrei
A
D002843:Chromatin / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D020552:Comet Assay; D004247:DNA / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects / Q000528:radiation effects; D053938:DNA Fragmentation / Q000187:drug effects / Q000528:radiation effects; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004996:Ethidium / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D014965:X-Rays
D002843:Chromatin; D004247:DNA; D004996:Ethidium