³²P-postlabelling for the sensitive detection of DNA adducts.
32P-postlabelling is a technique originally described by Kurt Randerath and colleagues for the sensitive detection of damage produced in DNA by reactive chemicals or genotoxins. The procedure essentially entails the enzymatic digestion of DNA to nucleoside 3'-monophosphates which are then radioactively labelled using T4 polynucleotide kinase and [γ(32)P]-adenosine triphosphate. Adducted nucleoside-3'-5'-bisphosphates are then separated from their normal counterparts by thin layer chromatography. Prior to the development of the assay, quantification of DNA adducts was confined to studies that utilised compounds synthesised to be isotopically labelled with tritium or carbon-14. As such, these studies were limited to specific and recognised genotoxins that could be administered only in the laboratory to cultures or animals. With (32)P-postlabelling it was possible not only to determine DNA adduct induction by a relatively uncharacterised suspected carcinogen, but also following exposure to complex mixtures containing a multitude of known and unknown potential genotoxins. The small amount of DNA required to perform the (32)P-postlabelling assay also meant that human biomonitoring studies using readily obtainable tissues, such as lymphocytes, were possible. Using the standard (32)P-postlabelling method, it is possible to detect a single DNA adduct in 10(7) to 10(8) normal nucleotides. The subsequent development of several enhancement methods improved this detection rate to one adduct in 10(10) nucleotides. For these reasons, the (32)-postlabelling assay represents an extremely versatile and extremely sensitive method to detect and monitor DNA damage.
Department of Biochemistry and Cell Biology, Institute of Integrative Biology, University of Liverpool, Biosciences Building, Crown Street, Liverpool, L69 7ZB, UK. firstname.lastname@example.org
SourceMethods in molecular biology (Clifton, N.J.) 817: 2012 pg 183-206
Chromatography, Thin Layer
Pub Type(s)Journal Article