The 32P-postlabeling assay for DNA adducts

Nat Protoc. 2007;2(11):2772-81. doi: 10.1038/nprot.2007.394.

Abstract

32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • DNA / chemistry
  • DNA Adducts / analysis*
  • Isotope Labeling / methods*
  • Phosphorus Radioisotopes*

Substances

  • DNA Adducts
  • Phosphorus Radioisotopes
  • DNA