Quantitation of Apurinic/Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts.

ABSTRACT: The apurinic/apyrimidinic (AP) site is a common lesion of DNA damage. The levels of AP sites reported in the literature cover a wide range, which is primarily due to the artifactual generation or loss of AP sites during processing of the DNA. Herein, we have developed a method for quantitating AP sites with a largely reduced level of artifacts by derivatizing AP sites before DNA isolation. A rapid digestion of nuclear protein was performed to minimize enzymatic DNA repair, followed by direct derivatization of AP sites in the nuclear lysate with O-(pyridin-3-yl-methyl)hydroxylamine, yielding an oxime derivative that is stable through the subsequent DNA processing steps. Quantitation was done using highly selective and sensitive liquid chromatography-tandem mass spectrometry, with a limit of

SUBMITTER: Chen H 

PROVIDER: S-EPMC6718090 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature