Synthesis of fluorescently labeled alkylated DNA adduct standards and separation by capillary electrophoresis

DNA adducts are regarded as individual internal dosimeters for the exposure to chemical carcinogens. To date, the most sensitive method for DNA adduct analysis is the radioactive P-32-postlabeling method, which allows the det ection of one adduct in 10(10) unmodified nucleotides in mu g amounts of DN A. However, this technique suffers from disadvantages such as working with radioactive phosphorus and time-consuming chromatographic separation proced ures. In addition, the simultaneous detection of adducts from different cla sses of carcinogens in a DNA sample is difficult. In order to overcome thes e drawbacks, we are developing a new detection method, comprising fluoresce nce labeling of DNA adducts, capillary electrophoretic (CE) separation, and on-line detection by monitoring laser-induced fluorescence (LIF). So far, we have evaluated the separation power and the detection limit of CE with f luorescently labeled standard compounds such as unmodified nucleotides or a lkylated thymidines. For this purpose, we developed a universal method for labeling 5'-OH-mononucleosid-3'-dicyanoethyl-phosphates with fluorescent dy es based on the phosphoramidite technology for DNA synthesis. The separatio n of N3-methylated, N3-, O-2- and O-4-butylated thymidines from the unmodif ied nucleotide within a few minutes recommends CE-LIF as a powerful method for DNA adduct analysis.