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Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay / 华中科技大学学报(医学)(英德文版)
Journal of Huazhong University of Science and Technology (Medical Sciences) ; (6): 104-6, 2005.
Article in English | WPRIM | ID: wpr-634233
ABSTRACT
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 II (Exo III) digestion. With Exo III 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.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Binding Sites / Polymerase Chain Reaction / Rats, Sprague-Dawley / Receptors, Aryl Hydrocarbon / Cytochrome P-450 CYP1A1 / Cytosol / DNA-Binding Proteins / Exodeoxyribonucleases / Polychlorinated Dibenzodioxins / Liver Type of study: Diagnostic study Language: English Journal: Journal of Huazhong University of Science and Technology (Medical Sciences) Year: 2005 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Binding Sites / Polymerase Chain Reaction / Rats, Sprague-Dawley / Receptors, Aryl Hydrocarbon / Cytochrome P-450 CYP1A1 / Cytosol / DNA-Binding Proteins / Exodeoxyribonucleases / Polychlorinated Dibenzodioxins / Liver Type of study: Diagnostic study Language: English Journal: Journal of Huazhong University of Science and Technology (Medical Sciences) Year: 2005 Type: Article