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Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay
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摘要 A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD:AhR:DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution. A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD:AhR:DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.
作者 孙晞 徐顺清
出处 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2005年第1期104-106,共3页 华中科技大学学报(医学英德文版)
基金 This project was supported by grants from National Natu ral Science Foundation of China (No. 20107002,20377017).
关键词 aryl hydrocarbon receptor dioxin-responsive element exonuclease S1 nuclase PCR aryl hydrocarbon receptor dioxin-responsive element exonuclease Ⅲ S1 nuclase PCR
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参考文献10

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