A32Ppostlabeling

The 32P-postlabeling assay is a versatile method developed in 198177 for a variety of DNA damage types.78 This method is extremely sensitive with the limit of detection in the range of 1 adduct per 109-10 or even 1011 nucleotides. In this method, DNA is hydrolyzed to a mixture of normal and adducted nucleotides and the hydrophobic adducted nucleotides are either additionally extracted with butanol for enrichment or the normal nucleotides removed from the assay by digestion with nuclease P1. The mixture is then phosphorylated using [g32P] ATP and polynucleotide kinase, separated by TLC or HPLC and the adduct levels calculated from radioactivity counts. The choice of enrichment procedure is determined by the type of target DNA lesion. 32P-postlabeling is a relatively nonspecific assay and is generally applicable to unidentified lesions. Most adducts of very different origin may be monitored, e.g., adducts of PAHs, aromatic amines, small alkylating agents, mycotoxins, chemotherapeutic drugs, oxidative damage generators, and adducts from complex mixtures. Another advantage of this technique is the requirement for small amounts of sample DNA making it ideal for target tissue measurements. There are several limitations of 32P-postlabeling. The adduct identification is frequently not possible because this method does not yield structural adduct data. However, with the further improvements of separation procedures for the 32P-postlabeled adducts (capillary electrophoresis or capillary electrochromatography, CE or CEC), much better characterization is expected.13

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