Optimized methodology for product recovery following emulsion PCR: applications for amplification of aptamer libraries and other complex templates
Keywords:emulsion PCR, ePCR, droplet PCR, methods, aptamers, techniques
Bias and background issues make efficient amplification of complex template mixes such as aptamer and genomic DNA libraries via conventional PCR methods difficult; emulsion PCR is being increasingly used in such scenarios to circumvent these problems. However, before products generated via 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 via 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 via 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 via emulsion PCR.
Polz MF, Cavanaugh CM. Bias in Template-to-Product Ratios in Multitemplate PCR. Appl Env Microbiol. 1998;64:7.
Meyerhans A, Vartanian J-P, Wain-Hobson S. DNA recombination during PCR. Nucleic Acids Res. 1990;18:1687–91.
Musheev MU, Krylov SN. Selection of aptamers by systematic evolution of ligands by exponential enrichment: Addressing the polymerase chain reaction issue. Anal Chim Acta. 2006;564:91–6.
Tawfik DS, Griffiths’ AD. Man-made cell-like compartments for molecular evolution. Nat Biotechnol. 1998;16:5.
Williams R, Peisajovich SG, Miller OJ, Magdassi S, Tawfik DS, Griffiths AD. Amplification of complex gene libraries by emulsion PCR. Nat Methods. 2006;3:545–50.
Shao K, Ding W, Wang F, Li H, Ma D, Wang H. Emulsion PCR: A High Efficient Way of PCR Amplification of Random DNA Libraries in Aptamer Selection. Veitia RA, editor. PLoS ONE. 2011;6:e24910.
Kanagal-Shamanna R. Emulsion PCR: Techniques and Applications. In: Luthra R, Singh RR, Patel KP, editors. Clin Appl PCR [Internet]. New York, NY: Springer New York; 2016 [cited 2019 Jun 28]. p. 33–42. Available from: http://link.springer.com/10.1007/978-1-4939-3360-0_4
Witt M, Phung NL, Stalke A, Walter J-G, Stahl F, von Neuhoff N, et al. Comparing two conventional methods of emulsion PCR and optimizing of Tegosoft-based emulsion PCR. Eng Life Sci. 2017;17:953–8.
Schaerli Y, Wootton RC, Robinson T, Stein V, Dunsby C, Neil MAA, et al. Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets. Anal Chem. 2009;81:302–6.
Schütze T, Rubelt F, Repkow J, Greiner N, Erdmann VA, Lehrach H, et al. A streamlined protocol for emulsion polymerase chain reaction and subsequent purification. Anal Biochem. 2011;410:155–7.
Green LS, Jellinek D, Jenison R, Ostman A, Heldin C-H, Janjic N. Inhibitory DNA Ligands to Platelet-Derived Growth Factor B-Chain. Biochemistry. 1996;35:14413–24.
Willard HH, Smith GF. THE PERCHLORATES OF THE ALKALI AND ALKALINE EARTH METALS AND AMMONIUM. THEIR SOLUBILITY IN WATER AND OTHER SOLVENTS 1. J Am Chem Soc. 1923;45:286–97.
Abil Z, Ellefson JW, Gollihar JD, Watkins E, Ellington AD. Compartmentalized partnered replication for the directed evolution of genetic parts and circuits. Nat Protoc. 2017;12:2493–512.
Boulanger J, Muresan L, Tiemann-Boege I. Massively Parallel Haplotyping on Microscopic Beads for the High-Throughput Phase Analysis of Single Molecules. Orlando L, editor. PLoS ONE. 2012;7:e36064.
Diehl F, Li M, He Y, Kinzler KW, Vogelstein B, Dressman D. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods. 2006;3:551–9.
Chen WW, Balaj L, Liau LM, Samuels ML, Kotsopoulos SK, Maguire CA, et al. BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles. Mol Ther - Nucleic Acids. 2013;2:e109.
Castellanos-Rizaldos E, Milbury CA, Makrigiorgos GM. Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion. Korolev S, editor. PLoS ONE. 2012;7:e51362.
Wang Y, Lu J, Yang Q, Bai Y, Ge Q. An Emulsion Based Microarray Method to Detect the Toxin Genes of Toxin-Producing Organisms. Molecules. 2011;16:7365–76.
Lu J, Zhang X, Zhang R, Ge Q. MicroRNA heterogeneity in endometrial cancer cell lines revealed by deep sequencing. Oncol Lett. 2015;10:3457–65.
How to Cite
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).