A simpler, more robust method for the analysis of 8-oxoguanine in DNA
A simpler, more robust method for the analysis of 8-oxoguanine in DNA
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DOI：
10.1016/s0891-5849(00)00316-6
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发表时间：
2000-08-01
影响因子：
7.4
通讯作者：
Ames, BN
中科院分区：
文献类型：
--
作者：
Beckman, KB;Saljoughi, S;Ames, BN
氧化DNA碱基8-氧鸟嘌呤通常是通过酶切DNA到核苷，然后用高效液相色谱（HPLC）分离加合物8-氧脱氧鸟嘌呤来测定的。最近，围绕这种方法的有效性存在着巨大的争论，由此可以清楚地看出，在样品制备的许多阶段，天然碱被人为氧化为8-氧鸟嘌呤。因此，我们设计了一种替代传统酶切DNA的方案，它(i)限制了人工氧化的可能性，（ii）显著加快了分析速度，（iii）适度提高了分析的灵敏度，（iv）解决了有关核酸酶切DNA效率的批评。简而言之，我们利用大肠杆菌修复酶甲酰胺嘧啶（Fapy）糖基化酶将碱基8-氧鸟嘌呤从全长DNA中释放出来，然后通过超滤（10000 Da排除）将8-氧鸟嘌呤从高分子量分子中分离出来，并通过反相高效液相色谱分析碱基加合物。这种方法的优点包括：(i)快速去除样品中大约百万倍的未改变碱基的摩尔量，（ii）减少酶培养的长度和步骤数，（iii）消除高温培养，（iv）非常干净的色谱分离，(v)快速洗脱分析物和相应的更大的吞吐量。使用这种改进的方法，我们跟踪了8-氧鸟嘌呤在过氧化物处理的HeLa细胞DNA中的诱导，这一实验用传统方法证明是繁琐的。(C) 2000 Elsevier Science Inc.；
The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine. There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation. Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases. In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC. Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput. Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods. (C) 2000 Elsevier Science Inc.