Compartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complex libraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-free manner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion (ePCR) including sample recovery by DNA purification. Furthermore, no special laboratory equipment is needed and inexpensive components are used. Therefore, our flexible protocol allows ePCR to be readily implemented in daily routine experiments for a broad range of applications

Erdmann, V.

Glokler, J.

Greiner, N.

Konthur, Z.

Lehrach, H.

Repkow, J.

Rubelt, F.

Schutze, T.

Schütze, T.

Erdmann, V. A.

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A streamlined protocol for emulsion polymerase chain reaction and subsequent purification.

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A streamlined protocol for emulsion polymerase chain reaction and subsequent purification

Anal Biochem

Analytical Biochemistry 410 (2011) 155–157Contents lists available at ScienceDirectAnalytical Biochemistryjournal homepage: www.elsevier .com/locate /yabioNotes & TipsA streamlined protocol for emulsion polymerase chain reactionand subsequent purificationTatjana Schütze a,b, Florian Rubelt a,b, Julia Repkow a, Nicole Greiner a, Volker A. Erdmann b, Hans Lehrach a,Zoltán Konthur a, Jörn Glökler a,⇑a Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Ihnestrasse 63-73, 14195 Berlin, Germanyb Institute for Chemistry/Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germanya r t i c l e i n f oArticle history:Received 21 September 2010Received in revised form 12 November 2010Accepted 17 November 2010Available online 25 November 20100003-2697/$ - see front matter  2010 Elsevier Inc. Adoi:10.1016/j.ab.2010.11.029⇑ Corresponding author. Fax: +49 (0) 30 8413 1380E-mail address: gloekler@molgen.mpg.de (J. Glökle1 Abbreviations used: BSA, bovine serum albuminpolymerase chain reaction.a b s t r a c tCompartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complexlibraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-freemanner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion(ePCR) including sample recovery by DNA purification. Furthermore, no special laboratory equipment isneeded and inexpensive components are used. Therefore, our flexible protocol allows ePCR to be readilyimplemented in daily routine experiments for a broad range of applications. 2010 Elsevier Inc. All rights reserved.The usefulness of applying emulsion PCR (ePCR)1 is clearly dem-onstrated by its routine application in next-generation sequencingtechniques. Amplification of DNA samples with a high diversity, suchas genomic or cDNA libraries, or other complex mixtures of differentsequences by conventional PCR may be compromised due to threegeneral problems. Artifacts are generated by recombination betweenhomologous regions, which results in the formation of chimeric DNAmolecules [1]. Additionally, short fragments tend to be amplifiedmore efficiently in comparison to longer ones [2]. Also, highly di-verse nucleic acids may not be able to form perfect duplexes follow-ing denaturation steps in PCR [3].A water-in-oil emulsion allows many reactions to occur inde-pendently in a single reaction tube. Distinct reaction compart-ments such as micelles provide controlled conditions by simplephysical separation. Thereby each droplet can be regarded asequivalent to a separate test tube.The advantages of ePCR are inhibition of trans-priming of differ-ent fragments, no biased amplification due to unequal amplifica-tion rates, and the same conditions for all individual sequences/DNA templates are applied.Current applications are ePCR on microbeads used for next-gen-eration sequencing, such as the SOLID or 454 technology [2,4],diagnostic DNA amplifications like BEAMing [5], or selections byin vitro compartmentalization [6]. Additionally, ePCR can be em-ployed for the bias-free amplification of DNA libraries, e.g., cDNAsll rights reserved..r).; ePCR, emulsion PCR; PCR,and other artificial highly diverse nucleic acids used in SELEXexperiments. For many of these applications, the anchoring tobeads in emulsions is not required. In such cases, the emulsionsare usually broken by the addition of the highly volatile and flam-mable organic solvent diethyl ether followed by a precipitationstep to isolate the amplified DNA [2,7]. In order to simplify theePCR protocol (Fig. 1), we replaced the use of a sonifier or magneticstirrer for compartmentalization by vortexing and simplified theDNA purification process by omitting precipitation. Instead, weadapted the procedure to be compatible with commercially avail-able DNA cleanup kits.We could determine that a homogeneous emulsion comparableto that of Diehl et al. [5] is readily formed by vigorous vortexingafter 3 min (Supplementary Fig. S2). To show that our simplifiedePCR protocol works efficiently, we have first applied it to the ini-tial amplification of a cDNA library directly after reverse transcrip-tion. We demonstrated that the size distribution of DNA is similarto that in an open PCR (nonemulsified control); however, no shiftsoccur (Fig. 2a). Further, we show that ePCR enhances the quality ofamplification of a diversified nucleic acid SELEX library by reducingartifact formation (Fig. 2b). We demonstrate that the protocol re-ported here preserves the diversity and complexity in DNA li-braries in accordance with other ePCR amplification procedures.PCR mixtures in 50-ll aliquots (aqueous phase) were preparedon ice under the same conditions as open PCR, containing 2.5 llBSA (10 mg/ml, NEB, USA). As BSA content may depend on whichPCR kit is used, it is advisable to verify the required amount ofBSA by titration. Since the distribution of template DNA in micellesis in concordance with Poisson distribution, the total number ofDNA molecules should not exceed 109 copies. This ensures theFig.1. Streamlined procedure for ePCR. The conventional PCR mixture is supplied with an oil surfactant mixture and an emulsion is generated by vigorous vortexing.Following PCR, emulsion is broken by the addition of isobutanol and supplemented with binding buffer from the DNA cleanup kit. After a short centrifugation, the resultingorganic phase is removed and the remaining aqueous phase is subjected to DNA purification according to the kit protocol.relative fluorescence unit length [bp]ePCR, 18 c. ePCR, 21 c. open PCR, 18 c. open PCR, 21 c.1300 700 2000 7000500RCPnoislumERCPnepoM      1010 109 108 107 106 M     1010 109 108 107 106 MSize- 200 bp- 100 bp- 60 bpabFig.2. (a) Analysis of amplicon size distribution by Agilent Bioanalyzer (High Sensitivity DNA Kit) after 18 and 21 cycles of open PCR and ePCR amplification from a humancDNA preparation. ePCR shows comparable distribution curves between 18 and 21 amplification rounds, whereas in open PCR a distortion of the amplified cDNA is observed(arrows). For a better comparison, the obtained curves were overlaid. (b) Aberrant band formation using different amounts of synthetic DNA library as starting template.Arrow indicates correct template size. In contrast to ePCR, the open PCR produces significantly more artifacts as visualized by ethidium bromide on a 3% agarose gel.156 Notes & Tips / Anal. Biochem. 410 (2011) 155–157presence of one or merely a few templates per micelle, a prerequi-site for an unbiased amplification (Fig. 2b) [2,8]. The componentsfor the emulsion were combined according to Diehl et al. [5].Briefly, 300 ll of prechilled oil surfactant containing 73% Tegosoft(Evonik, Germany), 20% mineral oil (Sigma, Germany), and 7% ABILWE (Evonik) was added to the 50-ll aqueous phase. Water-in-oil-emulsions were generated by putting the samples on a standardVortex-Genie 2 vortexer (Scientific Industries Inc., USA) at fullspeed (2700 rpm) for 5 min at +10 C in a cold room. The resultingemulsion was divided into 100-ll aliquots in PCR cups and incu-bated for 15–20 PCR cycles in a thermal cycler. Denaturation tem-perature did not exceed 95 C.To break the emulsion, corresponding samples were pooled in a2-ml reaction cup and 1 ml isobutanol was added followed byshort vortexing until the sample clarifies (usually 5 s). From sev-eral tested organic solvents, isobutanol proved optimal for han-dling and efficiency (see Supplementary material). Followingaddition of DNA binding buffer from the DNA cleanup kit andNotes & Tips / Anal. Biochem. 410 (2011) 155–157 157subsequent vortexing, the organic and aqueous phases were easilyseparated by centrifugation for 1 min at 16,000g. The organic phasewas discarded and DNA was purified with a DNA cleanup kit fromthe remaining aqueous phase. Multiple DNA cleanup kits were val-idated and best results were obtained using the GeneMatrix PCR/DNA kit from EURx (Poland; purchased from Roboklon, Germany).Amplified DNA in the aqueous phase could also be purified withmagnetic beads using the Sbeadex magnetic beads from LGCGenomics (England) according to the manufacturers directions(data not shown).In order to monitor the correct amplification of DNA and to ver-ify whether the numbers of amplification cycles are sufficient, it isadvisable to use a small aliquot of an open PCR sample that wasrun in parallel reaction as a control in agarose gel electrophoresis.If the thermal stability of the emulsion during PCR needs to bechecked, we recommend emulsifying primers and template sepa-rately, and pooling the generated emulsions in a single PCR tube.Stability should result in the absence of PCR product in comparisonto the normal control setup.The described protocol is compatible with modernhigh-throughput techniques. By using commercially availablecomponents for purification, it can be easily combined with exist-ing automated processes. Next to the described examples of ePCRwith cDNA and SELEX libraries (Fig. 2), we envisage that our ePCRprotocol is applicable in many research areas and is not restrictedto mere DNA amplification (PCR). It is possible to integrate ePCRinto other protocols related to handling of diverse libraries suchas gDNA and cDNA amplification, haplotyping, directed molecularevolution, and selection processes, e.g., for optimizing properties ofnovel enzymes and more [9].AcknowledgmentsThis work was partially supported by the European Unionthrough the EFRE Program (ProFIT Grant, Contract No. 10139409)and by the German Ministry of Science and Education (BMBF,Grant 0315448B).Appendix A. Supplementary dataSupplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ab.2010.11.029.References[1] A. Meyerhans, J. Vartanian, S. Wain-Hobson, DNA recombination during PCR,Nucleic Acids Res. 18 (1990) 1687–1691.[2] R. Williams, S.G. Peisajovich, O.J. Miller, S. Magdassi, D.S. Tawfik, A.D. Griffiths,Amplification of complex gene libraries by emulsion PCR, Nat. Methods 3 (2006)545–550.[3] T. Schütze, M. Menger, A. Wochner, M. Vingron, V.A. Erdmann, H. Lehrach, et al.,A calibrated diversity assay for nucleic acid libraries using DiStRO – a diversitystandard of random oligonucleotides, Nucleic Acids Res. 38 (2010) e23.[4] Y. Schaerli, F. Hollfelder, The potential of microfluidic water-in-oil droplets inexperimental biology, Mol. BioSyst. 5 (2009) 1392.[5] F. Diehl, M. Li, Y. He, K.W. Kinzler, B. Vogelstein, D. Dressman, BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions, Nat. Methods 3(2006) 551–559.[6] D.S. Tawfik, A.D. Griffiths, Man-made cell-like compartments for molecularevolution, Nat. Biotechnol. 16 (1998) 652–656.[7] Q. Ge, Z. Liu, Y. Bai, D. Zhang, P. Yu, Z. Lu, Emulsion PCR-based method to detectY chromosome microdeletions, Anal. Biochem. 367 (2007) 173–178.[8] A. Swami, G. Espinosa, S. Guillot, E. Raspaud, F. Boué, D. Langevin, Confinementof DNA in water-in-oil microemulsions, Langmuir 24 (2008) 11828–11833.[9] A.D. Griffiths, D.S. Tawfik, Miniaturising the laboratory in emulsion droplets,Trends Biotechnol. 24 (2006) 395–402.

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