Improvements in the 32P-postlabelling procedure to quantify bulky aromatic DNA adducts

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.