A sensitive biochemical assay for the detection of uracil
A sensitive biochemical assay for the detection of uracil
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DOI：
10.1002/em.20165
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发表时间：
2006-01-01
影响因子：
2.8
通讯作者：
Heydari, AR
中科院分区：
文献类型：
--
作者：
Cabelof, DC;Nakamura, J;Heydari, AR
我们已经开发了一种灵敏的检测DNA中尿嘧啶的新方法。本文所述的测定法是对中村等人（中村等人[1998]：Cancer Res 58：222-225）开发的高灵敏度噬菌体狭缝印迹（ASB）测定法的改进，其中通过生物素化的噬菌体反应性探针与DNA的结合来检测噬菌体DNA损伤（ADL）。尿嘧啶DNA糖基化酶（UDG）偶联ASB检测试剂盒使用尿嘧啶-DNA糖基化酶生成脱碱基位点，随后通过ASB方法进行检测。修改该技术用于检测尿嘧啶的能力具有以下优点：需要少量DNA（4 μ g DNA）;该测定适用于来自细胞和组织的DNA;灵敏度与通过较难获得的方法如气相色谱-苔藓光谱法（GC-MS）获得的灵敏度一样好; DNA链断裂不是混杂变量;通过使用甲氧胺阻断预先存在的前列腺病变;变异非常低（
We have developed a sensitive new assay for the detection of uracil in DNA. The assay described here is an adaptation to the highly sensitive aldehydic slot blot (ASB) assay developed by Nakamura et al. (Nakamura et al. [1998]: Cancer Res 58:222-225) in which aldehydic DNA lesions (ADLs) are detected through binding of a biotinylated aldehydic reactive probe to DNA. The uracil DNA glycosylase (UDG)-coupled ASB assay uses uracil-DNA glycosylase to generate an abasic site, which is subsequently detected by the ASB methodology. The ability to modify this technique for the detection of uracil has these advantages: small quantities of DNA are required (4 mu g of DNA); the assay is adaptable to DNA from both cells and tissues; sensitivity is as good as that achieved by less accessible methodologies, like gas chromatography-moss spectroscopy (GC-MS); DNA strand breaks are not a confounding variable; preexisting aldehydic lesions are blocked through the use of methoxyamine; variation is very low (