The semi-quantitative comparison of oxidative stress mediated DNA single and double strand breaks using terminal deoxynucleotidyl transferase mediated end labeling combined with a slot blot technique.
The accumulation of DNA damages by environmental stresses is represented by the steady state level of single strand breaks (SSBs) and double strand breaks (DSBs). Terminal deoxynucleotidyl transferase (TdT) mediated end labeling is suitable in detecting DSBs, but is unsuitable for SSBs due to its catalyzing characteristics. However, the sensitivity of TdT to detect SSBs may be significantly improved by first denaturing the double strands and expose all the DNA nicks as potential substrates for TdT. By coupling DNA denaturation to slot blot southern hybridization, the authors demonstrate the sensitive detection of SSBs as well as DSBs in 20 ng DNA samples derived from a retinal pigment epithelial cell line treated with tert-butyl hydroperoxide. The signal intensity of denatured and TdT-treated DNA in slot blot hybridization correlated to the amount of SSBs calculated in an S1 nuclease digestion assay. The signal ratio between denatured and non-denatured DNA likely approximates the SSBs/DSBs ratio in genomic DNA. The combination of DNA denaturing, TdT treatment and slot blot hybridization could be a useful method to assess oxidative stress-induced DNA strand damages.
Department of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. sighonda@med.kobe-u.ac.jp
Shigeru
S
Honda
Iichiro
I
Sugita
Katsuaki
K
Miki
Isao
I
Saito
D004247:DNA / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000187:drug effects*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D012680:Sensitivity and Specificity; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D004277:DNA, Single-Stranded; D004247:DNA; D020122:tert-Butylhydroperoxide; D004253:DNA Nucleotidylexotransferase