Schneider Lindsay, Cui Francis, Tripathi Anubhav
Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, Rhode Island 02912, USA.
Biomicrofluidics. 2021 Mar 17;15(2):024104. doi: 10.1063/5.0045307. eCollection 2021 Mar.
The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina's sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.
诸如Illumina测序平台等新一代测序（NGS）技术的出现和传播，已使与DNA和RNA测序相关的成本、时间及技术难度大幅降低。尽管有此趋势，但为测序生成核酸文库所需的工作流程仍然耗时且费力。以下研究提出了一种方法，通过用一种整合了磁分离和电动纯化（MSEP）的新型微流控方法取代基于磁珠的常规清洗步骤，来简化和优化这一过程。MSEP无需泵、移液器混合、涡旋或离心，它依靠目标DNA选择性吸附到磁珠上，随后磁珠通过一个经历反平行电渗流的微通道进行传输。利用一个简单的电流体动力学流动模型对协同流动条件进行了优化。这项工作表明，MSEP在从连接后文库混合物中消除接头二聚体方面与手工方法同样有效，同时还大大减少了实际操作时间和所需的移液量。尽管目前MSEP已专门应用于NGS文库制备，但它有潜力被改编并用于任何基于磁珠的分离方案，即微尺度上的固相萃取、序列特异性杂交和免疫沉淀。
