Steenwinkel M J, Roggeband R, van Delft J H, Baan R A
Department of Genetic Toxicology, TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
IARC Sci Publ. 1993(124):65-70.
This contribution describes methodological modifications and improvements that may contribute to inter-assay reproducibility and more accurate adduct quantification for 32P-postlabelling. Firstly, an anion-exchange chromatography procedure was developed to determine the amount of DNA used per assay and to check its purity, in particular to verify the absence of contaminating RNA. Secondly, calibration standards were prepared, in order to correct for differences in recovery. The modification levels of these standards were determined by synchronous fluorescence spectrophotometric analysis. Thirdly, the effect on adduct levels of exposure to light during postlabelling was investigated. Exposure of polyaromatic DNA adducts on a PEI-cellulose plate reduced the amounts of adducts detected considerably.
本论文描述了一些方法学上的改进，这些改进有助于提高32P后标记法的批间重复性和更准确的加合物定量。首先，开发了一种阴离子交换色谱法，以确定每次检测所用DNA的量并检查其纯度，特别是验证是否不存在污染性RNA。其次，制备校准标准品，以校正回收率的差异。这些标准品的修饰水平通过同步荧光分光光度分析来确定。第三，研究了后标记过程中光照对加合物水平的影响。PEI纤维素板上多环芳烃DNA加合物的光照显著降低了检测到的加合物量。
