Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay.
- Author:
Xi, SUN
;
Shunqing, XU
- Publication Type:Journal Article
- MeSH:
Binding Sites;
Cytochrome P-450 CYP1A1/*genetics;
Cytosol/metabolism;
DNA-Binding Proteins/chemistry;
Exodeoxyribonucleases/chemistry;
Liver/*metabolism;
Polymerase Chain Reaction;
Rats, Sprague-Dawley;
Receptors, Aryl Hydrocarbon/*chemistry;
Tetrachlorodibenzodioxin/*analogs & derivatives;
Tetrachlorodibenzodioxin/chemistry
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2005;25(1):104-6
- CountryChina
- Language:English
-
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.
