Yindeeyoungyeon W, Schell M A
University of Georgia, Athens 30602-2605, USA.
Biotechniques. 2000 Nov;29(5):1034-6, 1038, 1040-1. doi: 10.2144/00295st05.
Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI Prism 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5' untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easy-to-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.
足迹法是研究DNA与蛋白质相互作用的一种重要工具。然而，它通常涉及昂贵、繁琐且危险的步骤，如放射性标记以及在聚丙烯酰胺测序凝胶上进行分析。我们开发了一种简单的四步足迹法，包括：（i）生成并纯化一个一端用6-羧基荧光素进行荧光标记的PCR片段；（ii）使该片段短暂暴露于一种DNA结合蛋白，然后再暴露于DNase I；（iii）通过旋转柱进行纯化；（iv）在ABI Prism 310毛细管DNA测序仪/基因分析仪上分析部分消化产物。如我们使用该方法来表征LysR型激活剂PhcA与它的一个受调控靶基因xpsR的5'非翻译区中两个相距超过100 bp的位点的结合情况所示，能够轻松获得非常详细且灵敏的大片段（> 400 bp）DNA足迹。这种新方法的优点在于：（i）使用寿命长、安全且易于制备的荧光标记靶片段；（ii）使用自动化DNA测序仪进行灵敏、可靠且高度可重复的片段分析，而非凝胶电泳和放射自显影；（iii）具有成本效益。
