Berezovski Maxim V, Musheev Michael U, Drabovich Andrei P, Jitkova Julia V, Krylov Sergey N
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Nat Protoc. 2006;1(3):1359-69. doi: 10.1038/nprot.2006.200.
Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers--a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.
适体通常通过指数富集配体系统进化技术（SELEX）从随机DNA（或RNA）序列文库中筛选得到，该技术涉及几轮交替进行的候选寡核苷酸分区和PCR扩增步骤。在此，我们描述一种非SELEX适体筛选方案——该过程涉及重复的分区步骤，且分区步骤之间无扩增。平衡混合物的非平衡毛细管电泳（NECEEM）是一种高效的亲和方法，用于分区。NECEEM还便于在分区的每个步骤监测富集文库的整体亲和力，并筛选单个克隆与靶标的亲和力。NECEEM允许在测序前对所有克隆进行筛选，以便仅对具有合适结合参数的克隆进行测序。整个方案可在1周内完成，而传统的SELEX方案即使在专业工业设施中也需要数周时间。
