A P-32 POSTLABELING ASSAY FOR THE DETECTION OF ALKYLPHOSPHOTRIESTERS IN DNA

Within the group of DNA alkylation products, phosphotriesters (PTE) are among the most stable lesions. Hence, alkyl PTE are attractive biomarkers for DNA alkylation monitoring purposes, We have developed a P-32-postlabelling method for the analysis of both methyl and ethyl PTE in DNA, Since PTE bonds are not cleaved by any known DNA degrading enzyme, they are easily obtainable as PTE dinucleoside monophospates. A purification step, separating the PTE dinucleoside monophosphates from interfering compounds, such as mono- or oligonucleotides resulting from incomplete digestion of DNA, was developed using Waters C-18 Sep-Pak cartridges, Phosphotriester dinucleoside monophosphates themselves are not a substrate for phosphorylation by polynucleotide kinase, Polynucleotide kinase probably requires a negative charge on the phosphate closest to the 5'-end, Therefore, prior to the postlabelling step they have to be converted into either phosphodiester dinucleoside monophosphates or 3'-phosphate alkylated mononucleotides by treatment with alkali. For analysis of the labelled compounds we developed a two-step procedure, combining TLC and HPLC, that gave very straightforward information on the composition of the rather complex mixture. The detection limit is similar to 20 fmol PTE.