PULSED-FIELD GEL-ELECTROPHORETIC ANALYSIS OF DNA DOUBLE-STRAND BREAKSIN MAMMALIAN-CELLS USING PHOTOSTIMULABLE STORAGE PHOSPHOR IMAGING

Double-strand break (dsb) induction and repair was determined in the h uman colon carcinoma cell line clone A using pulsed-field gel electrop horesis (PFGE) coupled with photostimulable storage phosphor imaging t echnology. Because C-14-radioactivity was measured in a dried agarose gel following electrophoresis, no laborious processing of the gel, cut ting 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 e lectrophoresis was determined to be log-linear as as a function of dos e, and dsb rejoining after irradiation could be measured for exposures as low as 5 Gy. The kinetic parameters of dsb rejoining are independe nt of the initial dose delivered within experimental error over the ra nge of 5-20 Gy and complete after 4 h of recovery. The use of photosti mulable storage phosphor imaging allows the use of low levels of radio nuclide incorporation for DSB analysis in radiosensitive mammalian cel ls that would not be possible by other methods.