Separation of DNA Fragments Induced by Ionizing Irradiation Using a Graded-field Gel Electrophoresis

Abstract

A method is described that allows a separation of X-ray induced DNA fragments by graded-field gel electrophoresis. Synchronized G₁ and asynchronous CHO cells were embedded in agarose and irradiated with X-ray doses ranging from 1 to 100 Gy. Following proteolysis by sarcosine and proteinase K, electrophoresis was run for 49 h using graded electric fields with stepwise increasing field strength (0·6, 1·5, 3 and 9 V/cm). Since the molecular size of DNA able to migrate decreased with increasing voltage, each voltage step led to the generation of a distinct DNA band with the largest fragments in band 1, fragments of intermediate size in bands 2 and 3 and the smallest fragments in band 4. Using yeast chromosomal DNA as a reference, the molecular weight of eluted fragments was calculated to range from 1 to 10 Mbp. It could be shown that the fragment size was not the only criterion that discriminates migrating from non-migrating DNA. DNA fragments were found to be retained in the well by an unknown factor presumably associated with DNA conformation. This retention factor increased with increasing fragment size. Graded-field gel electrophoresis also allowed the determination of the absolute number of double-stranded breaks (dsb) induced, which amounted to 11·5 × 10⁻¹² dsbs Gy⁻¹ Da⁻¹ corresponding to 37 dsbs/G₁ cell.