MULTILESIONAL ASSAYS FOR OXIDATIVE DNA DAMAGE

提出了一种测量DNA氧化损伤的方法。 32P贴标程序的改编。该方法具有重要的意义 从损害赔偿是在 二聚体水平。二聚体方法是可能的，因为某些DNA损伤 可以通过使用以下方法从DNA中分离出修饰的二核苷酸单磷酸 核酸酶的适当组合。我们发现，通过使用 二核一磷酸，即使被氧化破坏，也是好的 T4多核苷酸激酶的底物；因此损伤二核苷酸 单磷酸盐可以选择性地用放射性磷标记。 这项拟议研究的目的是扩大化验范围，以包括 已知的几种DNA损伤是由氧化应激引起的。 实际上，我们设想了两种多层次的检测方法，一种是基于抑制的。 一种是核酸酶P1，另一种是抑制脾 磷酸二酯酶。这些DNA氧化损伤检测方法的一个特点是 航母的使用。介绍了一种载体，提取了关键 来自暴露在氧化应激下的细胞。对运营商的需求可能 阻碍了该分析在其他领域的广泛使用 实验室。讨论了克服这一障碍的方法。

A method of measuring oxidative DNA damage is presented which is an adaptation of the 32P post labeling procedure. The method has important advantages that derive from the fact that he damages are measured at the dimer level. The dimer approach is possible because certain DNA lesions can be isolated from DNA as modified dinucleotide monophosphates by using the appropriate combination of nucleases. We found that by using dinucleaside monophosphates, even though oxidatively damaged, are good substrates for T4 polynucleotide kinase; thus the damage dinucleotide monophosphates can be tagged selectively with radioactive phosphorous. The aim of the proposed research is to broaden the assay to include several lesions known to be induced in DNA by oxidative stress. Actually, two multi-lesional assays are envisaged, one base on inhibition of nuclease P1, the other based on inhibition of spleen phosphodiesterase. A feature of these assays of oxidative DNA damage is the use of carriers. Introduction of a carrier was the key extracted from cells exposed to oxidative stress. The need for carriers may present an impediment to widespread use of the assay in other laboratories. Ways of overcoming this impediment are discussed.