Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA.
We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.
Van Houten
B
B
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, TX, USA. vanhout1@niehs.nih.gov
Cheng
S
S
Chen
Y
Y
D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D018736:DNA Adducts / Q000032:analysis / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects*; D004272:DNA, Mitochondrial / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts; D005914:Globins / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D018401:Sample Size; D012680:Sensitivity and Specificity; D013539:Simian virus 40 / Q000235:genetics; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004272:DNA, Mitochondrial; D009838:Oligodeoxyribonucleotides; D005914:Globins; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D002945:Cisplatin