Immunofluorescence detection of radiation-induced DNA base damage.
We previously described a sensitive assay for measuring thymine glycol in the DNA of irradiated cells. The assay combines immunorecognition of the DNA lesion with capillary electrophoresis and laser-fluorescence detection to achieve an absolute detection level in the zeptomole (10(-21) mol) range. This article describes modifications to the protocol that overcome certain technical problems seen with the original methodology. In particular, the capillary electrophoresis is carried out at pH 8.3 rather than pH 10.5. The new protocol was used to examine removal of thymine glycol from the DNA of A549 lung adenocarcinoma cells and resting lymphocytes after exposure to 2 Gy gamma rays. Both cell types displayed similar repair kinetics. Removal of thymine glycol is almost complete at 4 hours postirradiation.
Weinfeld
Michael
M
Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2.
Xing
James Z
JZ
Lee
Jane
J
Leadon
Steven A
SA
Le
X Chris
XC
D000230:Adenocarcinoma / Q000235:genetics*; D004247:DNA / Q000528:radiation effects*; D019075:Electrophoresis, Capillary; D005455:Fluorescent Antibody Technique; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000235:genetics*; D013936:Thymidine / Q000031:analogs & derivatives* / Q000528:radiation effects
C029390:thymidine glycol; D004247:DNA; D013936:Thymidine