title: Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).
abstract: The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.
authors.affiliation: Department of Genetic and Cellular Toxicology, Merck Research Laboratories, WP45-320, West Point, PA 19486, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Laws
authors.forename: T R
authors.initials: TR
authors.lastname: Skopek
authors.forename: M V
authors.initials: MV
authors.lastname: Reddy
authors.forename: R D
authors.initials: RD
authors.lastname: Storer
authors.forename: W E
authors.initials: WE
authors.lastname: Glaab
mesh_terms: D000818:Animals; D001483:Base Sequence; D001711:Biotransformation; D002460:Cell Line; D018736:DNA Adducts / Q000032:analysis* / Q000235:genetics*; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects; D005456:Fluorescent Dyes; D005914:Globins / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D009152:Mutagenicity Tests; D009153:Mutagens / Q000493:pharmacokinetics / Q000633:toxicity; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D019914:Taq Polymerase
chemical_list: D018736:DNA Adducts; D017931:DNA Primers; D015342:DNA Probes; D005456:Fluorescent Dyes; D009153:Mutagens; D010761:Phosphorus Radioisotopes; D005914:Globins; D006861:Hydrogen Peroxide; D019914:Taq Polymerase