Single extraction protocol for the analysis of 8-hydroxy-2'-deoxyguanosine (oxo8dG) and the associated activity of 8-oxoguanine DNA glycosylase.
Determination of the promutagenic base 8-hydroxy-2'-deoxyguanosine (oxo(8)dG) has been the hallmark of studies aimed to determine oxidative damage to DNA. Different techniques including HPLC, GC-mass spectrometry, DNA sensitive sites and histology have been used to quantify oxo(8)dG levels in samples from different sources. The most accepted and well-established methods are based on HPLC and the ability of oxo(8)dG to be oxidized with an electrochemical detector. Considerable concerns have been raised in the ability of different labs to utilize a process of DNA extraction that reduces the levels of artifactual oxo(8)dG formed during sample workup. Here, we present a fully detailed protocol that has been extensively used in our Lab to extract and analyze DNA and has little or no impact in the basal levels of oxo(8)dG. Additionally, this protocol allows for the determination of the activity of mOgg1, the enzyme responsible for the initial step in the repair of the accumulated oxo(8)dG, in the same sample in which oxo(8)dG is detected.
Bolin
Celeste
C
Department of Pharmaceutical Sciences, Center for Environmental Health Sciences, University of Montana, Missoula, MT 59812, USA.
Stedeford
Todd
T
Cardozo-Pelaez
Fernando
F
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002540:Cerebral Cortex / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003497:Cyclic N-Oxides / Q000494:pharmacology; D045647:DNA Glycosylases / Q000032:analysis* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis*; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL
D003497:Cyclic N-Oxides; C024829:8-hydroxyguanine; D006147:Guanine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse; D003849:Deoxyguanosine; C003959:TEMPO