A modified alkaline comet assay for measuring DNA repair capacity in human populations.
Trzeciak, A. R., Barnes, J. and Evans, M. K. A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations. Radiat. Res. 169, 110-121 (2008). Use of the alkaline comet assay to assess DNA repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells (PBMCs), insufficient control of interexperimental variability, and limited analysis of DNA repair kinetics. We show that unstimulated cryopreserved PBMCs can be used in DNA repair studies performed using the comet assay. We have applied data standardization for the analysis of DNA repair capacity using negative and positive internal standards as controls for interexperimental variability. Our standardization procedure also uses negative controls, which provides a way to minimize the interference of interindividual variation in baseline DNA damage levels on DNA repair capacity measurements in populations. DNA repair capacity was assessed in a small human cohort using the parameters described in the literature including initial DNA damage, half-time of DNA repair, and residual DNA damage after 30 and 60 min. We have also introduced new DNA repair capacity parameter, initial rate of DNA repair. There was no difference in DNA repair capacity between fresh and cryopreserved PBMCs when measured by the Olive tail moment and tail DNA. The use of DNA repair capacity parameters in assessment of fast and slow single-strand break repair components is discussed.
Trzeciak
Andrzej R
AR
Laboratory of Cellular and Molecular Biology, and National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA.
Barnes
Janice
J
Evans
Michele K
MK
D002478:Cells, Cultured; D020552:Comet Assay / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism / Q000528:radiation effects
D004247:DNA