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

详细信息    查看全文

• 作者：John Ernest Vallarta Bajacan ; In Seok Hong ; Trevor W. Penning ; Marc M. Greenberg
• 刊名：Chemical Research in Toxicology
• 出版年：2014
• 出版时间：July 21, 2014
• 年：2014
• 卷：27
• 期：7
• 页码：1227-1235
• 全文大小：381K
• ISSN：1520-5010

文摘

8-Oxo-7,8-dihydro-2鈥?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.m>129m>, 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.