MULTILESIONAL ASSAYS FOR OXIDATIVE DNA DAMAGE

提出了一种测定DNA氧化损伤的方法， 32 P后标记程序的调整。 该方法具有重要的 由于损害赔偿额是按 二聚体水平。 二聚体方法是可能的，因为某些DNA损伤 可以从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 单磷酸双核苷酸，即使被氧化破坏， T4多核苷酸激酶的底物;因此， 可以用放射性磷选择性地标记单磷酸盐。 拟议研究的目的是扩大测定范围，包括 已知由氧化应激在DNA中诱导的几种损伤。 实际上，设想了两种多损伤测定，一种基于抑制 另一种是基于抑制脾脏的核酸酶P1 磷酸二酯酶 这些氧化性DNA损伤测定的特征是 使用载体。 引入载体是提取的关键 来自暴露于氧化应激的细胞。 对载体的需求可能 这对在其他领域广泛使用该测定法构成了障碍 laboratories. 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.