Blomstergren A, O'Meara D, Lukacs M, Uhlén M, Lundeberg J
Royal Institute of Technology (KTH), Stockholm, Sweden.
Biotechniques. 2000 Aug;29(2):352-4, 356, 358 passim. doi: 10.2144/00292rr03.
Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid co-purification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.
核酸杂交是当今许多标准分子生物学技术的重要组成部分。在最近的一项研究中，我们研究了能否通过利用堆积杂交来改善核酸捕获，堆积杂交是指寡核苷酸以连续串联方式杂交时存在的稳定效应。在此，我们描述了一种使用与单链靶标杂交的协同探针纯化测序产物的具体方法，其中一个探针已与磁珠偶联。这种方法是针对标准测序引物开发的，并已应用于鸟枪法质粒文库。协同探针被设计为在共同载体序列内退火，以避免未延伸的测序引物和错配测序产物的共纯化。磁珠的重复使用以及不依赖盐的洗脱，使得该方法适用于高容量毛细管电泳仪器。
