A modified neutral comet assay: elimination of lysis at high temperature and validation of the assay with anti-single-stranded DNA antibody.
Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion. In this report, we describe modifications of the neutral comet method, which simplify and facilitate its use for estimation of DNA DSB in X-irradiated mammalian cells in culture. The analysis carried out according to this protocol takes less time than those most often applied. Also, the use of lysis at 50 degrees C is avoided; this is important in view of the presence of heat-labile sites in the chromatin of irradiated cells, recently reported by Rydberg [Radiation-induced heat-labile sites that convert into DNA double-strand breaks, Radiation Research 153 (2000) 805-812]. The comets have well-defined, sharp limits, suitable for image analysis. The chromatin of the hydrogen peroxide-treated or UV-C-irradiated cell remains condensed similarly to that of the control cells. We checked the neutral comets for the presence of single-stranded DNA by means of a specific antibody. The results point to a satisfactory sensitivity of the modified neutral comet assay and its specificity for DSB. The minimum detection level of the modified neutral comet assay is about 5 Gy.
Wojewódzka
Maria
M
Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, PL-03-195, Warsaw, Poland. marlaw@orange.ichtj.waw.pl
Buraczewska
Iwona
I
Kruszewski
Marcin
M
D000818:Animals; D000974:Antibodies, Antinuclear; D016466:CHO Cells; D020552:Comet Assay / Q000379:methods*; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000276:immunology; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000633:toxicity; D006863:Hydrogen-Ion Concentration; D014466:Ultraviolet Rays / Q000009:adverse effects
D000974:Antibodies, Antinuclear; D004277:DNA, Single-Stranded; D004247:DNA; D006861:Hydrogen Peroxide