Lu Yue, Bianco Piero
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
J Biol Methods. 2021 Feb 24;8(1):e145. doi: 10.14440/jbm.2021.350. eCollection 2021.
Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70-15000 bp.
涉及DNA或RNA的单分子研究需要高质量的核酸底物均一制剂。在过去几年中，已经发表了多种描述不同纯化方法的文章，但即便由经验丰富的实验科学家操作，这些方法也存在令人沮丧的不一致性，产量也不稳定。为了解决这些问题，我们提出了一种优化且直接的基于柱的方法，该方法具有可重复性，能产生高质量的底物或底物组分，且产量高。所提出方法成功的关键在于使用无孔阴离子交换树脂。除了使用这种树脂外，我们鼓励在底物构建的每个步骤进行优化。完全优化后的方法能产生高质量的发夹DNA底物，且产量高。虽然这种底物适用于单分子磁镊实验，但所描述的方法很容易适用于大多数涉及大小从70至15000 bp的核酸的单分子研究中的DNA底物生产。
