A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' r egion of the MYC gene of cells of the human bladder carcinoma cell line, EJ -30, by using PCR with inverted primers. When the cells were allowed to rep air/misrepair for 6 or 23 h after irradiation, the frequency of rearrangeme nts increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for uni rradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(- 5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy, No significant dif ference was observed between 6 and 23 h of repair, Sequences obtained from the products suggest that there was no homology between the two sequences i nvolved in the recombination event and that there was no clustering of brea kpoints, The procedure is relatively simple, requiring only one digestion w ith a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located be tween a pair of primer sites 120 bp apart for which the primers face in opp osite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained, However, if an intrachromosomal rearrangement h as occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occu rred either within the same chromatid or between two sister chromatids, res pectively. Digestion between the primers would linearize an episome or rele ase a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be fa cing each other and would be located on either end of the linear molecule; and if they are less than similar to 5 kb apart, PCR amplification should r esult in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants, Also, quantification is based on the number of PCR products detect ed in a known amount of DNA, and not on a precise determination of the amou nt of PCR amplification that has occurred, Thus the inverse PCR procedure h as the potential of being used as an assay to detect variations in radiatio n-induced frequencies of DNA rearrangements. (C) 1999 by Radiation Research Society.