Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay.
The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.
Medical Biophysics Department, British Columbia Cancer Research Centre, 601 W. 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3.
J P
JP
Banáth
A
A
Kim
P L
PL
Olive
D000818:Animals; D002460:Cell Line / Q000528:radiation effects; D020552:Comet Assay / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis / Q000528:radiation effects*; D004249:DNA Damage; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000166:cytology; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors
D004247:DNA