Detection of platinum-DNA adducts by 32P-postlabelling.

Abstract:

: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 the assay was 1 adduct/10(7) nucleotides in a 10 micrograms DNA sample.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Blommaert FA,Saris CP

doi

10.1093/nar/23.8.1300

subject

Has Abstract

pub_date

1995-04-25 00:00:00

pages

1300-6

issue

8

eissn

0305-1048

issn

1362-4962

pii

5b0016

journal_volume

23

pub_type

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