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 chai n reaction (QPCR) methodology. Since the sensitivity of this assay is limit ed to the size of the DNA amplification fragment, conditions have been foun d for the quantitative generation of PCR fragments from human genomic DNA i n the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb pr oduct 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 be ta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to i ncreasing fluences of ultraviolet light (UV) resulted in the linear product ion of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesion s/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinet ic analysis of repair following 10 J/m(2) of WC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt ge ne and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies u sing 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. The se data indicate that the use of long targets in the gene-specific QPCR ass ay allows the measurement of biologically relevant lesion frequencies in 5- 30 ng of genomic DNA. This assay will be useful for the measurement of huma n exposure to genotoxic agents and the determination of human repair capaci ty. (C) 2000 Elsevier Science B.V. All rights reserved.