O'Connell Grant C, Smothers Christine G
School of Nursing, Case Western Reserve University, Cleveland, OH 44106-4904, USA.
J Biol Methods. 2020 Mar 10;7(1):e128. doi: 10.14440/jbm.2020.316. eCollection 2020.
Bias and background issues make efficient amplification of complex template mixes such as aptamer and genomic DNA libraries conventional PCR methods difficult; emulsion PCR is being increasingly used in such scenarios to circumvent these problems. However, before products generated emulsion PCR can be used in downstream workflows, they need to be recovered from the water-in-oil emulsion. Often, emulsions are broken following amplification using volatile organic solvents, and product is subsequently isolated precipitation. Unfortunately, the use of such solvents requires the implementation of special environmental controls, and the yield and purity of DNA isolated by precipitation can be highly variable. Here, we describe the optimization of a simple protocol which can be used to recover products following emulsion PCR using a 2-butanol extraction and subsequent DNA isolation a commercially available clean-up kit. This protocol avoids the use of volatile solvents and precipitation steps, and we demonstrate that it can be used to reliably recover DNA from water-in-oil emulsions with efficiencies as high as 90%. Furthermore, we illustrate the practical applicability of this protocol by demonstrating how it can be implemented to recover a complex random aptamer library following amplification emulsion PCR.
偏差和背景问题使得常规PCR方法难以有效扩增诸如适配体和基因组DNA文库等复杂模板混合物；在这种情况下，乳液PCR正越来越多地被用于规避这些问题。然而，在乳液PCR产生的产物可用于下游工作流程之前，需要将它们从油包水乳液中回收。通常，使用挥发性有机溶剂进行扩增后会打破乳液，随后通过沉淀分离产物。不幸的是，使用此类溶剂需要实施特殊的环境控制，并且通过沉淀分离的DNA的产量和纯度可能变化很大。在这里，我们描述了一种简单方案的优化，该方案可用于在乳液PCR后使用2-丁醇萃取并随后通过市售的纯化试剂盒分离DNA来回收产物。该方案避免了使用挥发性溶剂和沉淀步骤，并且我们证明它可用于从油包水乳液中可靠地回收DNA，效率高达90%。此外，我们通过展示如何实施该方案以在乳液PCR扩增后回收复杂的随机适配体文库来说明该方案的实际适用性。
