Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay |
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Authors: | Sun Xi Xu Shunqing |
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Affiliation: | 1. Department of Food Science and Engineering, Jinan University, Guangzhou 510060, China 2. Institute of Environmental Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430080, China |
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Abstract: | Summary 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 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, female, born in 1970, Lecturer, M. D., Ph. D.
This project was supported by grants from National Natural Science Foundation of China (No. 20107002, 20377017). |
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Keywords: | aryl hydrocarbon receptor dioxin-responsive element exonuclease III S1 nuclase PCR |
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