CONDITIONS INFLUENCING YIELD AND ANALYSIS OF 8-HYDROXY-2'-DEOXYGUANOSINE IN OXIDATIVELY DAMAGED DNA

被引：96

作者：

FLOYD, RA ^{[1
]}

WEST, MS ^{[1
]}

ENEFF, KL ^{[1
]}

SCHNEIDER, JE ^{[1
]}

WONG, PK ^{[1
]}

TINGEY, DT ^{[1
]}

HOGSETT, WE ^{[1
]}

机构：

[1] US EPA,CORVALLIS,OR 97330

来源：

ANALYTICAL BIOCHEMISTRY | 1990年 / 188卷 / 01期

关键词：

D O I：

10.1016/0003-2697(90)90544-J

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We have conducted studies to obtain practical knowledge regarding the stability, 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, we found that storage of the DNA at -20°C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20°C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100°C 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 causes 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. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG. © 1990.

引用

页码：155 / 158

页数：4

共 15 条

[1] RAPID ISOLATION OF CARCINOGEN-BOUND DNA AND RNA BY HYDROXYAPATITE CHROMATOGRAPHY [J].