Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay
- Author:
Xi SUN
1
;
Shunqing XU
Author Information
1. Jinan University
- Keywords:
aryl hydrocarbon receptor;
dioxin-responsive element;
exonuclease Ⅲ;
S1 nuclase;
PCR
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2005;25(1):104-106
- CountryChina
- Language:Chinese
-
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-tet rachlorodibenzo 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.
