Pulsed-field Gel Electrophoretic Analysis of DNA Double-strand Breaks in Mammalian Cells Using Photostimulable Storage Phosphor Imaging

Abstract

Double-strand break (dsb) induction and repair was determined in the human colon carcinoma cell line clone A using pulsed-field gel electrophoresis (PFGE) coupled with photostimulable storage phosphor imaging technology. Because ¹⁴C-radioactivity was measured in a dried agarose gel following electrophoresis, no laborious processing of the gel, cutting out regions of interest, liquid scintillation counting, etc., was necessary thereby saving labour, time and cost. The signal generated by phosphor screens was linear over 5 logs and sensitive to low levels of radionuclide exposure. Migration of broken DNA into the gel upon electrophoresis was determined to be log-linear as as a function of dose, and dsb rejoining after irradiation could be measured for exposures as low as 5 Gy. The kinetic parameters of dsb rejoining are independent of the initial dose delivered within experimental error over the range of 5–20 Gy and complete after 4h of recovery. The use of photostimulable storage phosphor imaging allows the use of low levels of radionuclide incorporation for DSB analysis in radiosensitive mammalian cells that would not be possible by other methods.