Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay / 华中科技大学学报(医学)(英德文版)
Journal of Huazhong University of Science and Technology (Medical Sciences)
;
(6): 104-106, 2005.
Artículo
en Chino
| WPRIM
| ID: wpr-336921
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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Tipo de estudio:
Estudio diagnóstico
Idioma:
Chino
Revista:
Journal of Huazhong University of Science and Technology (Medical Sciences)
Año:
2005
Tipo del documento:
Artículo
Similares
MEDLINE
...
LILACS
LIS