کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2148345 | 1089555 | 2011 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method](/preview/png/2148345.png)
Bulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical phenol-extraction procedure on the measurement of bulky DNA adducts by 32P-postlabelling, and to increase the throughput of the 32P-postlabelling method – whilst maintaining radio-safety – by reducing the radioisotope requirement per sample. The test DNA samples were prepared from MCF-7 cells treated with benzo[a]pyrene and from human peripheral blood lymphocytes, buffy coat, and peripheral lung tissue. The modified 32P-postlabelling procedure involved an evaporation-to-dryness step after the enzymatic digestions of the DNA, and radio-labelling with a reduced amount of [γ-32P]ATP substrate in a reduced reaction volume compared with the regular method. Higher levels of DNA adducts were measured in the MCF-7 cells and in the lung-tissue samples after isolation with the kit than after solvent extraction. A seven-fold higher level of adducts was detected in the buffy-coat DNA samples isolated with the kit than with the phenol extraction procedure (p < 0.001). Reduction of the amount of [γ-32P]ATP from 50 μCi to 25 μCi (>6000 Ci/mmol specific radioactivity) per sample in the modified 32P-postlabelling procedure was generally applicable without loss of adduct recovery for all test samples prepared with both DNA isolation methods. The difference between the bulky DNA-adduct levels resulting from the two DNA-isolation procedures requires further systematic investigation. The modified 32P-postlabelling procedure allows a 50% reduction of radioisotope requirement per sample, which facilitates increased throughput of the assay whilst maintaining radio-safety.
Journal: Mutation Research/Genetic Toxicology and Environmental Mutagenesis - Volume 721, Issue 1, 18 March 2011, Pages 95–100