一种适用于SELEX技术的单链DNA制备方法  

A Method to Generate Single-stranded DNA Through SELEX Technology

作　　者：刘中成[1] 解瑶[1] 张艳芬[2] 赵丽健 刘利鹏[1] 肖敏华[3] 

机构地区：[1]河北大学药学院,河北保定071002 [2]河北大学实验室管理办公室,河北保定071002 [3]河北大学附属医院肿瘤外科,河北保定071000

出　　处：《中国农业科技导报》2014年第2期149-154,共6页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(81202338);河北省自然科学基金项目(H2013201128;C2010000248);中国博士后基金项目(2013M530885)资助

摘　　要：为了建立一种简单、高效制备高纯度单链DNA方法,用于SELEX技术筛选过程中次级文库的筛选,对制备链霉亲和素磁珠的实验条件进行了优化,确定了对称PCR产物与链霉亲和素磁珠偶联的最佳时间和饱和度,并验证了不同浓度NaOH对解链效果的影响,建立了适于SELEX技术的单链DNA制备体系。利用荧光法和聚丙烯酰胺凝胶电泳对本实验所建立方法及传统不对称PCR法制备单链DNA的回收效率和纯度进行了比较,结果表明在最优实验条件下用磁性分离法制备的单链DNA纯度及回收效率均显著高于不对称PCR法。In order to establish a simple and effective method for preparing single-stranded DNA in SELEX screening process, this study optimized the experimental conditions for preparing the streptavidin-conjugated magnetic nanoparticles, decided the best time and saturation of symmetric PCR product binding with streptavidin-conjugated magnetic nanoparticles, verified the influence of different NaOH concentration on desmolysis effect, and established the ssDNA preparation system applied to SELEX technology. The purity and recovery efficiency of ssDNA obtained by established method in this experiment and traditional asymmetric PCR method was compared using fluorescence and polyacrylamide gel electrophoresis. The result showed that under optimal experimental condition the method established in this experiment was significantly better than asymmetric PCR method.

关 键 词：SELEX技术 单链DNA 链霉亲和素磁珠 不对称PCR 

分 类 号：Q5-33[生物学—生物化学]