一种新的酶切保护PCR分析方法及其在二恶英类化合物检测中的应用  

A Developmental Exonuclease Protection Mediated PCR Assay Applied to Detect the Dioxin-Like Chemicals

作　　者：孙晞[1] 李芳[1] 陈启政[1] 孙纳[1] 王小利[1] 严红[1] 徐顺清[1] 

机构地区：[1]华中科技大学同济医学院环境医学研究所,湖北武汉430030

出　　处：《癌变．畸变．突变》2004年第4期196-198,202,共4页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金资助(No.20107002;20377017)

摘　　要：背景与目的:建立一种灵敏、快速、无放射性污染的生物方法来检测环境样本中的二恶英类化合物。 材料与方法 :二恶英类化合物可以活化胞浆内芳香烃受体(AhR)使之与一段包含特定序列的双链DNA结合 ,结合的DNA因受蛋白质保护可抵抗核酸外切酶消化而保留下来 ,痕量的保留DNA通过PCR检测出来。根据此原理本研究将TCDD溶液加入到含AhR及相关蛋白的细胞溶质中 ,在体外与含二恶英反应元件的DNA作用形成二恶英_AhR_DNA复合物 ,用核酸外切酶ExoⅢ和S1核酸酶进行消化后作PCR ,通过琼脂糖电泳可以检测目的DNA是否存在 ;同时以有机溶剂DMSO设为对照。结果 :在实验组用琼脂糖电泳可以检测到期望大小片段的DNA,而对照组为阴性。结论 :该方法可作为环境样本中二恶英污染的生物检测手段 ,具有灵敏。BACKGROUND&AIM: For the non_radioactive,sensitive and rapid detection of the dioxin _ like chemicals in the environmental samples,exonuclease protection mediated PCR assay was established. MATERIAL AND METHˉODS: 2,3,7,8_ tetrachlorodibenzo_p_dioxin(TCDD)dissolved in DMSO,a typical and the most toxic ligand to transform AhR,was added into SD rat hepalic cytosol in vitro,which contained Ah receptors and relative proteins,and ligand_AhR_DRE complex are formed in addition of DNAs containing the sequence of DRE.With the digestion of ExonucleaseⅢand S1nuclease,free DNAs were digested to mononucleotide and bound DNA remained due to protein(AhR)protction and could be amplified by PCR.The effects of PCRs were shown by loading on2%agarose electrophoresis.DMSO was used as negative control was set up. RESULTS: Target DNA could be observed in the TCDD groups,but not in the control group. CONCLUSIONS: Exonuclease protection mediated PCR assay is a good non_radioactive tool to quantify the interaction of protein and DNA with high sensitivity and simple.

关 键 词：二恶英类化合物 芳香烃受体 二恶英反应元件 外切酶Ⅲ S1核酸酶 

分 类 号：R991[医药卫生—毒理学]