适体SELEX筛选中单链DNA的制备

摘要

探讨建立适体SELEX筛选中单链DNA(ssDNA)的制备方法,生成适体筛选的次级库,为适体SELEX筛选奠定基础.将PCR扩增后带生物素标记的双链DNA(dsDNA)连接在链霉亲和素标记的凝胶上,用NaOH变性解链制备ssDNA.利用荧光法分析dsDNA与凝胶结合所需的时间及NaOH对生物素与链霉亲和素标记凝胶结合的影响,在优化的试验条件下制备ssDNA,用琼脂糖和聚丙烯酰胺凝胶电泳对其产物进行鉴定.结果显示:生物素标记的dsDNA与链霉亲和素标记的凝胶结合的时间以40 min为佳,0.01 mol/L、0.10 mol/L和1.00 mol/L的NaOH均不影响生物素与链霉亲和素标记凝胶的结合;在优化试验条件下制备的产物为ssDNA.表明采用上述方法制备ssDNA,操作简单、方便,所需时间短,适用于适体SELEX筛选中次级库的生成.%This study aimed to find a easy way to prepare ssDNA and therefore lay a foundation for the construction of secondary ssDNA library for aptamer screening by systematic evolution of ligand by exponential enrichment (SELEX). PCR technique was employed, and SDS-PAGE analysis was applied to identify the product. The results showed that the optimized binding time of the double stranded DNA labeled with biotin and gel labeled with streptavidin were 40 min, and the binding of biotin and gel was independent of the concentration of NaOH. SDS-PAGE revealed that the product prepared under optimized condition was ssDNA.