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

Within the group of DNA alkylation products, phosphotriesters (PTE) ar e among the most stable lesions. Hence, alkyl PTE are attractive bioma rkers 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 enzym e, they are easily obtainable as PTE dinucleoside monophospates. A pur ification step, separating the PTE dinucleoside monophosphates from in terfering compounds, such as mono- or oligonucleotides resulting from incomplete digestion of DNA, was developed using Waters C-18 Sep-Pak c artridges, Phosphotriester dinucleoside monophosphates themselves are not a substrate for phosphorylation by polynucleotide kinase, Polynucl eotide kinase probably requires a negative charge on the phosphate clo sest to the 5'-end, Therefore, prior to the postlabelling step they ha ve to be converted into either phosphodiester dinucleoside monophospha tes or 3'-phosphate alkylated mononucleotides by treatment with alkali . For analysis of the labelled compounds we developed a two-step proce dure, combining TLC and HPLC, that gave very straightforward informati on on the composition of the rather complex mixture. The detection lim it is similar to 20 fmol PTE.