Detection of DNA strand breaks using PCR amplification for irradiated plasmid and its application for analysis of complex structure of DNA.

Abstract

Detection of DNA strand breaks by radiation was carried out using real-time PCR amplification. The method is able to target to given sequences. Gamma irradiation to pBR322 plasmid solutions was done by 137Cs, gammacell 40, as dose from 0 to 250Gy. 1000bp, 750bp, 500bp and 250bp fragments were amplified by combined primer sets to irradiated pBR322 plasmid DNA solution including 0~2mM Tris. The results were compared with simulation calculation and then the condition of p=0.2 was more fit to actual data for linear plasmid. The data from DNA solution containing closed circular (CC)plasmids was not fit between data from PCR and simulation.When this closed circular shape might be very difficult to be proceeded by PCR reaction, predicted calculation was fit to actual data of PCR. Therefore, we might estimate contents of CC DNA in the given plasmid solution. [J Radiat Res 44:436 (2003)]