Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).
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.
Laws
G M
GM
Department of Genetic and Cellular Toxicology, Merck Research Laboratories, WP45-320, West Point, PA 19486, USA.
Skopek
T R
TR
Reddy
M V
MV
Storer
R D
RD
Glaab
W E
WE
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
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