P-32-postlabeling assay for the quantification of the major platinum-DNA adducts

To allow more sensitive, selective, and routine analyses of platinum(Pt)-GG and -AG intrastrand crosslinks we have significantly improved our quantita tive P-32-postlabeling assay (M. J. P. Welters et al. Carcinogenesis 18, 17 67-1774, 1997), Instead of off-line scintillation counting we introduced an on-line flow radioisotope detector into the HPLC system. Furthermore, the isolation protocol for the adducts was significantly modified and optimized to reduce interfering background peaks that prevented quantification of lo w levels of the cisplatin-DNA adducts in white blood cells obtained from pa tients, Reduction of background signals was obtained by boiling the samples , followed by phenol/chloroform/isoamylethanol extraction after dine DNA di gestion step. The labeling efficiency for the adducts was increased by 40% by using Na-formate instead of NH4-formate for elution of the adducts from the strong cation-exchange columns. Finally, a calibration curve and qualit y controls were implemented, The labeling efficiencies were not different b etween the dinucleotides, The between- and within-run precision for the Pt- GG and PL-AG; adducts measured at the lower limit of quantification of 87 a nd 53 amol/mu g DNA, respectively, was less than 20% CV. The adducts were s table in DNA stored for a a-month time period at -80 degrees C, The assay i s now routinely used for high-precision analyses of patient and cell line s amples containing very low adduct levels. (C) less Academic Press.