改造地高辛标记DNA和检测试剂盒用于凝胶阻滞实验的新方法  

A New Method for EMSA by Modifying DIG High Prime DNA Labeling and Detection Starter Kit Ⅱ

作　　者：祁小廷[1] 柴小清[2] 刘靖[2] 柴团耀[1] 

机构地区：[1]中国科学院研究生院生物系,北京100049 [2]首都师范大学生命科学学院,北京100037

出　　处：《遗传》2006年第6期721-725,共5页Hereditas（Beijing)

基　　金：国家自然科学基金项目资助(编号:39870078;30370128;30570146)~~

摘　　要：凝胶阻滞实验(gel retardation),又称为电泳迁移率变动实验(Electrophoretic mobility shift assay,EMSA),是研究蛋白和DNA相互作用的一种技术。传统的32P标记探针的凝胶阻滞实验,具有很高的灵敏度,然而也有接触危险性的放射性同位素且不容易定量分析的缺点。最近利用非放射性标记的凝胶阻滞实验,已有很多成功的报道,该方法快捷,安全,灵活,但非放射性标记探针的凝胶阻滞试剂盒的费用却很高。在论文中,我们提供了一种改造地高辛标记DNA和检测试剂盒用于凝胶阻滞实验的新方法。首先将双链DNA探针末端引入EcoRⅠ粘性末端以便进行3′末端标记,然后利用价格比较便宜的地高辛标记DNA和检测试剂盒(DIG High Prime DNA Labeling andDetection Starter KitⅡ,Rohe)进行探针标记和凝胶阻滞信号检测。经过多次实验参数的摸索,最终得到了成功的结果,为利用地高辛标记DNA和检测试剂盒进行凝胶阻滞实验提供了成功的例子和方法。Gel retardation, also named electrophoretic mobility shift assay （EMSA） , is a useful tool for identifying protein-DNA interactions. Typically, 32 P-labeled DNA probes used in EMSA are sensitive. However, it relies on the handling of hazardous radioisotopes, and is not easily quantified. Recently, some successful cases have been reported using non-radio labelled probes instead of radiolabelled probes in EMSA. The method is rapid, convenient, and safe, but it depends on a very expensive kit. in this study, we offered a new method performing EMSA by modifying DiG High Prime DNA Labeling and Detection Starter Kit Ⅱ （Rohe）. Firstly, the prepared labeled probe was introduced the EcoR Ⅰ stick in the end of probe for 3＇-end labeling, and then was performed the probe labeling and detecting the signals of EMSA with the relatively cheap DIG High Prime DNA Labeling and Detection Starter Kit Ⅱ （Rohe）. By adjusting the experiment parameters, the successful result was obtained. The present study provides a successful example and method for modifying DIG High Prime DNA Labeling and Detection Starter Kit Ⅱ.

关 键 词：凝胶阻滞实验 地高辛 探针 

分 类 号：Q78[生物学—分子生物学]