title: Quantitative detection of 8-Oxo-7,8-dihydro-2'-deoxyguanosine using chemical tagging and qPCR.
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.
authors.affiliation: Department of Chemistry, Johns Hopkins University , 3400 N. Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: John Ernest Vallarta
authors.initials: JE
authors.lastname: Bajacan
authors.forename: In Seok
authors.initials: IS
authors.lastname: Hong
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Penning
authors.forename: Trevor W
authors.initials: TW
authors.lastname: Penning
authors.forename: Marc M
authors.initials: MM
authors.lastname: Greenberg
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D001205:Ascorbic Acid / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D019809:Streptavidin / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D004220:Disulfides; D005296:Ferrous Compounds; D016159:Tumor Suppressor Protein p53; D001710:Biotin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D019809:Streptavidin; D006861:Hydrogen Peroxide; D003849:Deoxyguanosine; D001205:Ascorbic Acid; C029451:ferrous chloride