Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay.
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.
Sun
Xi
X
Department of Food Science and Engineering, Jinan University, Guangzhou 510060, China.
Xu
Shunqing
S
D000818:Animals; D001665:Binding Sites; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry; D008099:Liver / Q000378:metabolism*; D008297:Male; D000072317:Polychlorinated Dibenzodioxins / Q000031:analogs & derivatives* / Q000737:chemistry; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry*
D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; C445379:1,2,7,8-tetrachlorodibenzo-p-dioxin; D019363:Cytochrome P-450 CYP1A1; D005090:Exodeoxyribonucleases