A method of measuring oxidative DNA damage is presented which is an adaptation of the 32P post labeling procedure. The method has important advantages that derive from the fact that he damages are measured at the dimer level. The dimer approach is possible because certain DNA lesions can be isolated from DNA as modified dinucleotide monophosphates by using the appropriate combination of nucleases. We found that by using dinucleaside monophosphates, even though oxidatively damaged, are good substrates for T4 polynucleotide kinase; thus the damage dinucleotide monophosphates can be tagged selectively with radioactive phosphorous.
The aim of the proposed research is to broaden the assay to include several lesions known to be induced in DNA by oxidative stress. Actually, two multi-lesional assays are envisaged, one base on inhibition of nuclease P1, the other based on inhibition of spleen phosphodiesterase. A feature of these assays of oxidative DNA damage is the use of carriers. Introduction of a carrier was the key extracted from cells exposed to oxidative stress. The need for carriers may present an impediment to widespread use of the assay in other laboratories. Ways of overcoming this impediment are discussed.
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