DETECTION AND SEQUENCING OF IONIZING RADIATION-INDUCED DNA REARRANGEMENTS USING THE INVERSE POLYMERASE-CHAIN-REACTION

Purpose: To develop a procedure, using the inverse polymerase chain re action, to detect and sequence ionizing radiation-induced DNA rearrang ements without prior phenotypic selection of mutant cells. Method: Nor mal human fibroblast cells (IMR-90) were given 30 Gy of gamma-irradiat ion and then incubated at 37 degrees C for 23 h to allow DNA repair. R earrangements of the sequence 5' to the c-myc gene were examined by am plifying the region using inverse PCR followed by DNA sequencing. Resu lts: Approximately fivefold more PCR products were amplified from the DNA of cells given 30 Gy of gamma-irradiation and allowed 23 h for rep air than were obtained from cells that were either unirradiated or wer e irradiated and then lysed immediately. PCR products from seven putat ive radiation-induced DNA rearrangements were sequenced. Of these prod ucts, one contained an unidentified sequence (a possible inter-chromos omal rearrangement) whilst the other products appeared to derive from episomes or duplication events (possible intra-chromosomal rearrangeme nts). The sequencing data suggested that the sites of DNA rearrangemen t breakpoints were non-randomly distributed and possibly associated wi th topoisomerase I consensus cleavage sequences. There was a significa nt level of direct homology between the sequences flanking the breakpo ints. Conclusions: The procedure developed was able to detect both int er- and intra-chromosomal rearrangements.