酶切保护聚合酶链反应检测多环芳烃受体与特异性DNA的结合

Detection of interaction of binding affinity of aromati c hydrocarbon receptor to the specific DNA by exonuclease protection polymerase chain reaction assay

目的 建立一种改进的核酸外切酶保护聚合酶链反应 (PCR)检测DNA 蛋白质结合物。方法 将 1pmol/L至 10nmol/L二英溶液 (TCDD)与 100μl含不同浓度多环芳烃受体的SD大鼠肝细胞溶质温育,再与 1fmol至 100nmol含二英反应元件的寡核苷酸探针作用形成复合物,用核酸外切酶Ⅲ和S1核酸酶进行消化后进行PCR,通过琼脂糖电泳检测PCR产物。同时设阴性对照(二甲基甲砜,DMSO)和空白对照。结果 在实验组用琼脂糖电泳可以检测到期望大小片段 (285bp)的DNA, 而对照组均为阴性。在一定范围类TCDD浓度与结合DNA之间存在剂量 效应关系。检测到的最小受体量为 2. 5fmol,最小DNA量为 2fmol。结论 核酸外切酶保护PCR无放射性污染,灵敏度和特异性高,简便易行,可作为DNA 蛋白质结合物的定性检测方法。

Objective To establish an ex onuclease protection mediated polymerase chain reaction (PCR) assay for the non -radioactive, sensitive detection of the binding of protein and DNA. Methods The 1 pmol/L-10 nmol/L 2,3,7,8- tetrachlorodibenzo-p-d ioxin (TCDD) dissolved in dimethyl sulphoxidey (DMSO), was added into 100 μl SD rat hepalic cytosol in vitro, which contained different amount of aromatic hydeocarbon receptors (AhR) and relative proteins, and ligand-AhR-DRE complex were formed in addition to 1 fmol/L-100 nmol/L DNAs probes containing the seque nce of DRE.With the digestion of Exonuclease Ⅲ and S1 nuclease, free DNAs wer e digested to oligonucleotide and binding DNA remained due to protein(AhR) prote ction and be amplified by PCR.The results of PCRs were shown by loading on 2% agarose electrophoresis.DMSO was used as negative control and blank control wa s set up.Results Target DNA(285bp) could be observed i n the ligand groups, but not in the control group.The minimal amount of recept or was 2.5 fmol/L and the minimal amount of DNA probes was 2 foml.C onclusions Exonuclease protection mediated PCR assay should be a go od non-radioactive tool to quantify the interaction of protein and DNA with hig h sensitivity and simplicity.