Quantitative polymerase chain reaction assay for DNA repair within defined genomic regions.
We have developed a quantitative assay to determine repair of structurally different DNA lesions at defined genomic sites. This assay depends on the fact that many different types of damage are repaired by the same nucleotide excision repair (NER) pathway which includes synthesis of short DNA fragments at the sites of damage. After exposure to damaging agents, cells are treated with 5-bromodeoxyuridine (BrdUrd) to label the regions undergoing repair with the presumption that regions that have been more efficiently repaired would incorporate more BrdUrd than regions that were less effectively repaired. Thus, the abundance of the different sequences in the BrdUrd-containing DNA would be a direct and quantitative measure for the repair rates of the corresponding regions. The BrdUrd-containing, repaired DNA was isolated by CsCl gradient centrifugation and immunoprecipitation with anti-BrdUrd antibody and was used as template in quantitative PCR in which the amount of the product was directly proportional to the amount of template. This approach was used to address the question whether DNA repair after UV-irradiation occurs in an uniform, random manner or with preferences for certain regions. We found out that there was a higher repair efficiency at the 5'-end of the mouse gamma-globin domain in Ehrlich ascites tumor cells.
Chakalova
L
L
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
Russev
G
G
D000818:Animals; D001973:Bromodeoxyuridine / Q000276:immunology / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair; D005796:Genes / Q000235:genetics; D016678:Genome; D005914:Globins / Q000235:genetics; D051379:Mice; D005810:Multigene Family / Q000235:genetics; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D012107:Research Design; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D005914:Globins; D004247:DNA; D001973:Bromodeoxyuridine