quantification of the drug incorporated in the DNA structure is in the center of attention. In this study we investigated the behavior of the platinum-based cytostatic drug and DNA adducts during the well-established Sanger sequencing method involving capillary electrophoretic (CE) separation. Three selected platinum-based cytostatic drugs (cisplatin, carboplatin and oxaliplatin) were incubated with the DNA fragment to create adducts and subsequently sequenced.

cytostatic drug. In Fig. 3, the analysis of carboplatinated DNA is shown. The signal of non-platinated DNA (control) is taken as 100%. In Fig. 3A, the signal of thymine was evaluated showing 50% decrease for 50 µg/ml of applied carboplatin. The signal decreased linearly with the concentration and it is shown that when 150 µg/ml of carboplatin is applied, the measured signal is only 6.3% of the control signal. Signal decrease was observed in case of all four bases; however the decrease was 36.5% (Fig. 3B), 31.4% (Fig. 3C) and 13.2% (Fig. 3D) in case of guanine, cytosine and adenine, respectively (when 50 µg/ml of carboplatin is applied). From the results can be concluded that cytostatics bound to the DNA influence the LIF signal for all four bases the same way which suggests that it is not affecting the CE-LIF analysis but the labeling reaction and the fragment synthesis by polymerase. We believe that when the drug is bound to the DNA the polymerase stops the synthesis of the complementary fragment and therefore the signal decreases. To verify the hypothesis, DNA adducts with cisplatin and oxalipaltin were prepared and analyzed by the same means. We observed that the concentration of the drug causing the same level of signal decrease differed for each type of cytostatic drug. To compare the results we calculated the concentration of the drug causing the decrease for 75% of the for control signal. It was found that for carboplatin 75 µg/ml is required. In case of oxalipaltin only 7 µg/ml is needed and finally only 0.3 µg/ml of cisplatin is causing such decrease. From these results it can be concluded that different cytostatics have different abilities to form adducts with DNA.