Storage phosphor imaging technique for detection and quantitation of DNA adducts measured by the 32P-postlabeling assay.
The 32P-postlabeling method has found wide application as a sensitive technique for detecting the presence of a broad range of bulky aromatic compounds covalently bound to DNA. In this method, the modified DNA is enzymatically degraded to 3'-mononucleotides and labeled with [32P]-phosphate at the 5'-position using [gamma-32P]ATP and T4 polynucleotide kinase. The 32P-labeled DNA digest is then chromatographed in two dimensions on polyethyleneimine - cellulose thin-layer plates. Screen-enhanced autoradiography is used to locate the presence of the radiolabeled adducts on the chromatogram, and the radioactive areas are generally excised and quantitated by liquid scintillation spectrometry. However, on a chromatogram with multiple adducts, it can be difficult to quantitative partially resolved adducts and evaluated background radioactivity levels. We have evaluated the use of storage phosphor imaging techniques to quantitate and map the radioactivity on chromatograms generated by the 32P-postlabeling method. The results showed that storage phosphor imaging was approximately 10 times more sensitive than screen-enhanced autoradiography at -80 degrees C for the detection of 32P, exhibits a greater linear range of response, has a resolution that compares favorably to film and has a lower background than does liquid scintillation spectrometry. Further, the generation of a digitized record of the distribution and intensity of radioactivity allows for computer-assisted assessment of adduct profiles and can facilitate quantitation of individual adducts and radioactive zones comprised of multiple overlapping adducts in complex chromatograms. Additionally, the permanent record created by the imaging technology permits facile retrospective analysis of samples, whereas with autoradiography and liquid scintillation spectrometry reanalysis of a replicate sample is required.
Reichert
W L
WL
Environmental Conservation Division, National Oceanic and Atmospheric Administration, Seattle, WA 98112-2097.
Stein
J E
JE
French
B
B
Goodwin
P
P
Varanasi
U
U
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D005399:Fishes; D010761:Phosphorus Radioisotopes; D013997:Time Factors
D010761:Phosphorus Radioisotopes; D004247:DNA