DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

Int J Radiat Biol. 1994 Jun;65(6):623-30. doi: 10.1080/09553009414550731.

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) x 10(-3)/Mbp/Gy was deduced for 80 kV X-rays.

MeSH terms

  • DNA / radiation effects*
  • DNA Damage*
  • DNA Probes
  • DNA, Complementary / genetics
  • Dose-Response Relationship, Radiation
  • Electrophoresis, Gel, Pulsed-Field
  • Humans
  • Lactoferrin / genetics*
  • Restriction Mapping
  • X-Rays

Substances

  • DNA Probes
  • DNA, Complementary
  • DNA
  • Lactoferrin