尼龙膜上DNA的化学发光检测
Chemiluminescent detection of DNA on nylon membranes.
作者信息
Dubitsky A, Brown J, Brandwein H
机构信息
Services Department, Pall Corporation, Glen Cove, NY 11542.
出版信息
Biotechniques. 1992 Sep;13(3):392-400.
Two types of nonradioactive DNA detection systems were optimized for use with nylon membranes in Southern transfers. A luminol substrate system (consisting of an enhanced chemiluminescent reaction utilizing luminol enzyme substrate) was used with peroxidase-labeled probe DNA, and a dioxetane-based substrate was used with alkaline phosphatase/antibody and digoxigenin-labeled probe DNA. Chemiluminescence was detected by autoradiography. Methods for reprobing the membranes were also optimized for both systems; blots could be reprobed at least ten times. Results showed that excellent sensitivity and low background can be achieved on both amphoteric and positively charged nylon membranes, using either detection system.
两种非放射性DNA检测系统针对Southern杂交中尼龙膜的使用进行了优化。一种鲁米诺底物系统（由利用鲁米诺酶底物的增强化学发光反应组成）与过氧化物酶标记的探针DNA一起使用，一种基于二氧杂环丁烷的底物与碱性磷酸酶/抗体及地高辛标记的探针DNA一起使用。通过放射自显影检测化学发光。两种系统的膜重杂交方法也进行了优化；印迹至少可重杂交十次。结果表明，使用任何一种检测系统，在两性和带正电荷的尼龙膜上都能获得出色的灵敏度和低背景。
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