Quantitative Detection of 8‑Oxo-7,8-dihydro-2′-deoxyguanosine Using Chemical Tagging and qPCR

ABSTRACT: 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-ox-odGuo) is a commonly formed DNA lesion that is useful as a biomarker for oxidative stress. Although methods for selective quantification of 8-oxodGuo exist, there is room for additional methods that are sensitive and utilize instrumenta-tion that is widely available. We previously took advantage of the reported reactivity of 8-oxodGuo to develop a method for detecting the lesion by selectively covalently tagging it with a molecule equipped with a biotin label that can be used subsequently with a reporting method (Xue, L., and Greenberg, M. M. (2007) J. Am. Chem. Soc. 129, 7010). We now report a method that can detect as little as 14 amol of 8-oxodGuo by tagging DNA with a reagent containing a disulfide that reduces background due to nonspecific binding. The reagent also contains biotin that enables capturing target DNA on streptavidin-coated magnetic beads. The captured DNA is quantified using quantitative PCR. The method is validated by comparing the amount of 8-oxodGuo detected as a function of Fe2+/H2O2/ascorbate-dose to that reported previously using mass spectrometry. DNA lesions can be mutagenic and have been implicated in