Detection of DNA strand breaks using real-time PCR amplification for irradiated plasmid pBR322 and its structural analysis

Abstract

Detection of DNA strand breaks by radiation was carried out using real-time PCR amplification. Gamma irradiation to pBR322 plasmid DNA solutions was done by 137Cs, gammacell 40, as dose of 0Gy, 30Gy, 60Gy, 100Gy, 150Gy, 200Gy and 250Gy. 1003bp, 749bp, 505bp and 242bp fragments were amplified by combined primer sets for the plasmid solution including Tris-HCl as a radical scavenger. Higher amplification values such as over 200% to the non-irradiated solution (100%) were observed and could be depending on rate of closed circular shape (CC) and open circular shape (OC) in the original solution of pBR322 used in each experiment. One suitable explanation that the CC is difficult to be amplified by PCR and after nicking by radicals, the reaction is progressed, was fit to the simulation pattern. Therefore, we carried out the PCR amplification for 13 irradiation points between 0~150Gy and the evident peak was observed at 30Gy. The results of the simulation were not completely fit to the PCR data. The explanation that all CC DNA could not be amplified might be not clear. We also tried to analyze whether the plasmid structure would be depending on the sequences or not. We will present the PCR data for the pBR322 exchanged with other similar length sequences.