Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses
Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses
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DOI：
10.1016/s0891-5849(01)00681-5
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发表时间：
2001-12-01
影响因子：
7.4
通讯作者：
Frenkel, K
中科院分区：
文献类型：
--
作者：
Huang, X;Powell, J;Frenkel, K
DNA中8-oxo-2'-脱氧鸟苷（8-oxo-dG）的估计值使用不同的定量方法甚至相同的方法至少相差一个数量级。我们的假设是，传统的核酸酶P1 （NP1）和碱性磷酸酶（AP）消化系统对DNA不完全水解成核苷起着重要的作用，导致HPLC结合UV和电化学（EC）检测结果的变异性。我们在这里表明，诸如DNA的数量、酶的选择、酶的活性和孵育时间等因素可以影响DNA的消化，从而导致8-o - dg水平的变化。在NP1 + AP系统中添加DNase I和磷酸二酯酶I和磷酸二酯酶I和磷酸二酯酶II，通过完全释放正常核苷和8-oxo-dG来改善DNA消化，从而减少8-oxo-dG水平的日间变化。二乙烯三胺五乙酸（DTPA）是一种铁螯合剂，在DNA消化过程中，以及在HPLC分析一批DNA样品时自动进样器的等待期间，可以防止8-oxo-dG的背景增加。在优化DNA消化条件后，使用市售鲑鱼睾丸DNA （ST DNA）测量8-oxo-dG的日间变异性在2年内为26%。在这些最佳条件下，我们的实验室变异性可能只占吸烟者群体氧化DNA损伤评估的总变异性的13%。基于我们的结果，我们相信改进的DNA消化条件将提供更准确的8-氧- dg测定，从而更可靠地估计癌症风险。(C) 2001爱思唯尔科学公司
Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing, to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers, Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk. (C) 2001 Elsevier Science Inc.