DNA Double-strand Break Measurement in Mammalian Cells by Pulsed-field Gel Electrophoresis: An Approach Using Restriction Enzymes and Gene Probing

Abstract

DNA samples prepared from human SP₃ cells, which had been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6·3 ± 0·7) × 10⁻³/Mbp/Gy was deduced for 80 kV X-rays.