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  • Title: 32P-Postlabeling/polyacrylamide gel electrophoresis analysis: application to the detection of DNA adducts.
    Author: Terashima I, Suzuki N, Shibutani S.
    Journal: Chem Res Toxicol; 2002 Mar; 15(3):305-11. PubMed ID: 11896676.
    Abstract:
    32P-Postlabeling analysis is a powerful technique to detect DNA adducts. Polyethylenimine-cellulose TLC plates are generally used to separate (32)P-labeled adducts using several different buffers. However, separation by TLC is time-consuming and labor-intensive for a large number of DNA samples. To expedite analyses, nondenaturing 30% polyacrylamide gel electrophoresis (PAGE) has been adapted for the (32)P-postlabeling analysis. The major advantages of this technique are as follows: (a) many DNA samples can be loaded concomitantly on the PAGE with standard markers; (b) DNA adducts can be resolved in only a few hours; and (c) exposure to (32)P during handing can be minimized. To show the usefulness of (32)P-postlabeling/PAGE analysis, the formation of a tamoxifen (TAM)-DNA adduct resulting from O-sulfonation of alpha-hydroxytamoxifen was demonstrated. In addition, to quantify TAM adducts, oligodeoxynucleotides containing diastereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen can be used as standards. The detection limit of this assay for 5 microg of DNA was approximately 7 adducts/10(9) nucleotides. The (32)P-postlabeling/PAGE analysis can also be used to detect DNA adducts derived from benzo[a]pyrene diol epoxide, 2-acetylaminofluorene, and 4-hydroxyequilenin.
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