Conditions Influencing Yield and Analysis of 8-Hydroxy-2' -Deoxyguanosine inOxidatively Damaged DNA

摘要

Studies have been conducted to obtain practical knowledge regarding thestability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxyguanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus UV light to form oxidatively damaged DNA, the authors found that storage of the DNA at -20C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100C for at least 15 min. Formation of 8-OHdG within DNA using UV light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers cause a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. (Copyright (c) 1990 Academic Press, Inc.)