Combining capillary electrophoresis and next-generation sequencing for aptamer selection.
Combining capillary electrophoresis and next-generation sequencing for aptamer selection.
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DOI：
10.1007/s00216-014-8427-y
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发表时间：
2015-02
影响因子：
4.3
通讯作者：
Bonin, Keith
中科院分区：
文献类型：
--
作者：
Riley, Kathryn R.;Gagliano, Jason;Xiao, Jiajie;Libby, Kara;Saito, Shingo;Yu, Guo;Cubicciotti, Roger;Macosko, Jed;Colyer, Christa L.;Guthold, Martin;Bonin, Keith
关键词：
下一代测序(NGS)机器可以在一次运行中对数百万条DNA链进行测序，例如由数百万到数万亿个离散寡核苷酸序列组成的寡核苷酸(OGO)库。毛细管电泳法是一种有吸引力的选择紧密结合寡核苷酸或“适配子”的技术，因为它需要最少的样本量(例如100毫升)，并提供了一个溶液相选择环境，通过该环境可以定量地测定靶向结合寡核苷酸的丰度。在这里，我们描述了使用基于毛细管瞬时等速电泳法(CtITP)的非平衡混合毛细管电泳法(NECEEM)作为一种从包含已知(29聚体)凝血酶结合适配子的随机文库中选择适配子的实验。我们的毛细管电泳法(CE)选择的样本被离子激流个人基因组机(PGM)测序，并分析了选择效率。我们表明，单轮CE选择可以使随机合成的DNA寡核苷酸混合物的凝血酶结合活性从选择前的0.4%适配子含量丰富到>；15%适配子含量。本文的在线版本(doi：10.1007/s00216-0148427-y)包含补充材料，授权用户可以使用。
Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or “aptamers” because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4 % aptamer content before selection to >15 % aptamer content. The online version of this article (doi:10.1007/s00216-014-8427-y) contains supplementary material, which is available to authorized users.
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