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基于凝胶阵列的序列特异性DNA结合蛋白荧光检测方法

发表时间:2017-12-15  浏览量:576  下载量:92
全部作者: 潘沁,陆祖宏,何农跃
作者单位: 中国药科大学生命科学与技术学院;东南大学生物科学与医学工程学院
摘 要: 聚丙烯酰胺凝胶阵列用于检测序列特异性DNA结合蛋白,具有高度并行性、微量化、高结合容量和低背景的优点。研究将通用寡核苷酸固定在聚丙烯酰胺凝胶阵列上形成单分子发夹寡核苷酸,通过固相延伸为双链寡核苷酸后,采用酶切法非标记检测序列特异性DNA结合蛋白NF-κB芯片扫描信号和荧光显微信号。经过4次平行检测实验,结果表明,与常用的基于玻璃片基检测荧光标记蛋白的方法相比,本研究采用的方法的灵敏度提高了2个数量级,并且具有高灵敏度和高特异性,为蛋白质与序列特异性DNA的亲和力等芯片研究提供了有力工具。
关 键 词: 生物药物学;非标记序列特异性DNA结合蛋白质;单分子发夹寡核苷酸;聚丙烯酰胺凝胶阵列
Title: A gel pad matrix-based approach for fluorometric detection of sequence specific DNA binding protein
Author: PAN Qin, LU Zuhong, HE Nongyue
Organization: Life Science and Technology School, China Pharmaceutical University; School of Biological Sciences and Medical Engineering, Southeast University
Abstract: Polyacrylamide gel pad (PGP) matrix shows high throughout, miniaturization, high binding content and low background for detection of sequence specific DNA binding protein. Herein, constant oligonucleotides were immobilized into PGP matrix and linked to unimolecular hairpin oligonucleotides. Then double strand DNA oligonucleotides were formed by elongation on the solid phase before labeled-free NF-κB was detected by cut of EcoR I in scanner and fluorescence microscopy. Compared with constant glass-based method for fluorescence labelled protein detection, the sensitivity of this method was increased in two orders of magnitude after 4 parallel testing experiments. The results indicated that this method showed high sensitivity and specificity and could provide a powerful tool for research with biochips on affinity between protein and sequence specific DNA.
Key words: biopharmaceutics; free-labeled sequence specific DNA binding protein; unimolecular hairpin oligonucleotides; polyacrylamide gel pad matrix
发表期数: 2017年12月第23期
引用格式: 潘沁,陆祖宏,何农跃. 基于凝胶阵列的序列特异性DNA结合蛋白荧光检测方法[J]. 中国科技论文在线精品论文,2017,10(23):2634-2640.
 
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