Kow Y W, Dare A
Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Avenue, Atlanta, Georgia 30335, USA.
Methods. 2000 Oct;22(2):164-9. doi: 10.1006/meth.2000.1057.
Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites. Many procedures are available for the quantification and detection of base damage and AP sites. However, either these procedures are laborious or the starting materials are difficult to obtain. A biotinylated aldehyde-specific reagent, ARP, has been shown to react specifically with the aldehyde group present in AP sites, resulting in biotin-tagged AP sites in DNA. The biotin-tagged AP sites can then be determined colorimetrically with an ELISA-like assay, using avidin/biotin-conjugated horseradish peroxidase as the indicator enzyme. The ARP assay is thus a simple, rapid, and sensitive method for the detection of AP sites in DNA. Furthermore, removal of damaged base by DNA N-glycosylases generates AP sites that can be measured by the ARP reagent. By coupling the ARP assay with either endonuclease III from Escherichia coli or 8-oxoguanine N-glycosylase (OGG1) from yeast, investigators can rapidly determine the amount of oxidative pyrimidine damage (endonuclease III-sensitive sites) or purine damage (OGG1-sensitive sites) in cellular DNA, respectively. An increased level of oxidative damage has been implicated in several age-related human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as the aging process. The sensitivity and simplicity of the ARP assay thus make it a valuable method for investigators who are interested in estimating the level of oxidative DNA damage in cells and tissues derived from patients with various age-related diseases or cancers.
活性氧会造成广泛的DNA损伤，包括氧化性碱基损伤和无碱基（AP）位点。有许多方法可用于定量和检测碱基损伤及AP位点。然而，这些方法要么繁琐，要么起始材料难以获得。一种生物素化的醛特异性试剂ARP，已被证明能与AP位点中存在的醛基特异性反应，从而在DNA中产生生物素标记的AP位点。然后，可以使用抗生物素蛋白/生物素偶联的辣根过氧化物酶作为指示酶，通过类似酶联免疫吸附测定（ELISA）的方法比色测定生物素标记的AP位点。因此，ARP测定法是一种检测DNA中AP位点的简单、快速且灵敏的方法。此外，DNA N-糖基化酶去除受损碱基会产生可被ARP试剂测量的AP位点。通过将ARP测定法与来自大肠杆菌的核酸内切酶III或来自酵母的8-氧代鸟嘌呤N-糖基化酶（OGG1）相结合，研究人员可以分别快速测定细胞DNA中氧化性嘧啶损伤（核酸内切酶III敏感位点）或嘌呤损伤（OGG1敏感位点）的量。氧化性损伤水平的升高与几种与年龄相关的人类疾病有关，如阿尔茨海默病、肌萎缩侧索硬化症和帕金森病，以及衰老过程。因此，ARP测定法的灵敏度和简便性使其成为对估计患有各种与年龄相关疾病或癌症的患者来源的细胞和组织中氧化性DNA损伤水平感兴趣的研究人员的一种有价值的方法。
