随机单链DNA文库SELEX筛选寡核苷酸适配子方法的建立

指数富集配基的系统进化(SELEX)技术是一种新的组合化学技术.体外构建了一个长度为81 nt、含有35个随机序列的单链DNA(ssDNA)文库，优化了ssDNA文库扩增为双链DNA (dsDNA)文库的PCR反应条件.通过对比不对称PCR和生物素-链亲和素磁珠分离方法制备ssDNA文库的效果，确定了以生物素-链亲和素磁珠分离方法制备ssDNA.由于脱氧核糖核酸的疏水性导致ssDNA文库与硝酸纤维素滤膜的结合背景过高，因此选择以微孔板为介质，分离与靶蛋白结合的适配子.经过9轮循环筛选，随机ssDNA文库与丙型肝炎病毒(HCV)核心蛋白(C蛋白)的结合率从0.5%上升到32.5%.

A Procedure for SELEX Screening Aptamers From ssDNA Random Library

SELEX(systematic evolution of ligands by exponential enrichment)is a new combinatorial chemistry technology.An 81 nucleotides ssDNA pool containing 35 random nucleotides flanked by invariant primer was designed.The PCR amplification conditions were optimized for converting the ssDAN pool into dsDAN pool. Compared to asymmetry PCR,the technique based on biotin-streptavidin-magnetism bead was suitable forgeneration of ssDAN from dsDAN.Since the hydrophobicity of deoxyribose leads to an inherently higher degree of background binding to nitrocellulose filters,panning procedures on microtiter plates were employed.After 9 rounds selection,the percentage of the ssDAN pool bound to HCV core protein inceased from 0.5% to 32.5%.