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.
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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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Xi, S., Shunqing, X. Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay. Current Medical Science 25, 104–106 (2005). https://doi.org/10.1007/BF02831401
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DOI: https://doi.org/10.1007/BF02831401