Title: Quantitative detection of 8-Oxo-7,8-dihydro-2'-deoxyguanosine using chemical tagging and qPCR.
Authors: Bajacan, John Ernest Vallarta; Hong, In Seok; Penning, Trevor M; Penning, Trevor W; Greenberg, Marc M
Published In Chem Res Toxicol, (2014 Jul 21)
Abstract: 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) 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 instrumentation 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 Fe(2+)/H2O2/ascorbate-dose to that reported previously using mass spectrometry.
PubMed ID: 24932862
MeSH Terms: Ascorbic Acid/chemistry; Biotin/chemistry; Deoxyguanosine/analogs & derivatives*; Deoxyguanosine/analysis; Deoxyguanosine/chemistry; Disulfides/chemistry; Ferrous Compounds/chemistry; Hydrogen Peroxide/chemistry; Plasmids; Polymerase Chain Reaction; Streptavidin/chemistry; Tumor Suppressor Protein p53/genetics