32P-postlabeling DNA damage assays: PAGE, TLC, and HPLC

32P-Postlabeling analysis is a powerful technique for detecting, identifying, and quantifying DNA adducts induced by mutagens or carcinogens. The method involves enzymatic digestion of the DNA sample to nucleoside 3'-monophosphates, and partial purification of the adducted nucleotides followed by their 5'-labeling with 32P. For analysis of DNA adducts, polyethyleneimine-cellulose thin-layer chromatography (TLC) plates have traditionally been used to resolve 32P-labeled DNA adducts (32P-postlabeling/ TLC analysis). However, the TLC procedure is time consuming and labor intensive. To expedite analyses, we recently devised a 32P-postlabeling protocol that utilizes nondenaturing polyacrylamide gel electrophoresis (PAGE) and permits multiple DNA samples to be run on a single gel (32P-postlabeling/PAGE analysis). Using this method, the detection limit for 5 microg of DNA is approx 7 adducts/10(9) nucleotides, similar to that for 32P-postlabeling/TLC. For still higher sensitivity and resolution, high-performance liquid chromatography (HPLC) combined with a radioisotope detector system (32P-postlabeling/HPLC analysis) can be used to increase the detection limit to approx 3 adducts/10(10) nucleotides. Here we describe all three 32P-postlabeling techniques.