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Nucleic Acids Research, 1995, Vol. 23, No. 8 1300-1306
© 1995

METHODS

Detection of platinum-DNA adducts by 32P-postlabelling

Frank A. Blommeart and Chris P. Saris*

Division of Molecular Carcinogenesis, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Huis) Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

* To whom correspondence should be addressed c/o R. Bernands

Received January 23, 1995. Revised March 10, 1995. Accepted March 10, 1995.

We developed a sensitive 32P-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography.Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of theassay was 1 adduct/107 nucleotides in a 10 µg DNA sample.


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