The alkaline elution technique for measuring DNA single strand breaks: increased reliability and sensitivity.
The alkaline elution procedure is noted, on the one hand, for its sensitivity in the detection of DNA damage, but on the other hand, for its extreme variability and inconsistency. These deficiencies in the technique have been traced to incomplete exchanges of the various solutions used in the cell rinse and lysis and DNA rinse and elution portions of the procedure. Solutions to the above indicated problems involve several changes to the standard procedure, including, at the cellular stage, the complete removal of rinsing solutions followed by the addition of lysis solution at 0 degrees C. After standard cell lysis and alkaline rinse of the DNA, the alkaline rinse solution is replaced by elution solution at high flow rate to allow a uniform starting time for the elution, which then proceeds as a nearly first-order function of time (not elution volume). Using gamma-rays as the damaging agent, reproducibility within and between experiments is easily comparable to radiation survival itself, and typically, duplicate elution channels within an experiment provide nearly identical information without the need for internal standards and complex ratiometric analysis techniques. The procedures described allow the reproducible assessment of radiation damage to cellular DNA at doses much below 1 Gy and allow the study of repair processes down to similar levels of residual damage. This unexpected increase in technique sensitivity may be caused by maintenance of near-freezing temperatures during cell manipulation and addition of lysis solutions or to more complete and uniform lysis.
Koch
C J
CJ
Department of Radiation Oncology, University of Pennsylvania, Philadelphia 19104-6072.
Giandomenico
A R
AR
D000468:Alkalies; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000468:Alkalies; D004277:DNA, Single-Stranded