Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells.
A new method is described for detecting DNA double-strand breaks (DSBs) that utilizes asymmetric field inversion gel electrophoresis (AFIGE). DNA purified from cells in agarose plugs is subjected to AFIGE and DNA breakage quantitated by the fraction of DNA released from the plug. To test the specificity of the method for DNA DSBs, purified DNA in agarose plugs was treated for increasing times with restriction endonuclease, XhoI. After an initial time period, the fraction of DNA released increased in direct proportion to time. This correlates with the expected response for a randomly broken DNA molecule. In contrast, treatment with the single-strand breaking agent, hydrogen peroxide, over a 1000-fold range produced no release of DNA from the plug. Thus the assay appears to be specific for DNA DSBs and was used to measure DNA breaks induced by gamma radiation. Purified DNA, irradiated in agarose plugs, exhibited a log-linear dose response up to doses that release greater than 90% DNA from the plug. When live cells were irradiated in agarose, a similar linear dose response was observed up to 40 Gy and a significant signal as low as 2.5 Gy. Also in live cells, a threefold lower percentage of DNA was released from the plug over the same dose range. However, less DNA per gray is released at doses above 40 Gy and may reflect a crosslinking effect produced by the irradiation of DNA in live cells. DNA which was "pulse-labeled" was used to test the effect of DNA replication on the ability of AFIGE to detect DNA DSBs. Replicating DNA irradiated in the cell or after purification exhibited a reduced rate of release from the plug per dose of irradiation. Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.
Stamato
T D
TD
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Denko
N
N
D000818:Animals; D002460:Cell Line; D002588:Cesium Radioisotopes; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000528:radiation effects*; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005720:Gamma Rays; D066298:In Vitro Techniques; D008920:Misonidazole / Q000494:pharmacology
D002588:Cesium Radioisotopes; D008920:Misonidazole; D004247:DNA