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BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions

Nature Methods volume 3, pages 551–559 (2006)Cite this article

The most important biotechnological advances made in the 20th century involved methods that converted single DNA molecules into populations of identical DNA molecules. The first wave of techniques used cells (cloning)1 and the second used PCR2. Cloning was advantageous because the populations arising from individual molecules were inherently separated. In contrast, each template required individual compartments (tubes) for PCR-based methods if separate products were desired. Emulsion PCR overcame this disadvantage by miniaturizing the compartments so that millions of templates could be individually amplified within a single tube3. BEAMing (beads, emulsions, amplification and magnetics) is a process built on emulsion PCR that (i) includes beads within the compartments and (ii) ensures that one strand of the PCR product is bound to the beads4. After amplification, each compartment contains a bead that is coated with thousands of copies of the single DNA molecule originally present. These beads can be recovered with a magnet or by centrifugation. Beads obtained via BEAMing accurately reflect the DNA diversity present in template populations and this method can be used to determine what fraction of a DNA population contains a specific mutation5. Because each bead contains thousands of molecules of the identical sequence, the signal to noise ratio obtained by hybridization or enzymatic assays is extremely high. Millions of beads can be analyzed within minutes using flow cytometry or optical scanning instruments. The DNA bound to beads also provides excellent templates for high-throughput sequencing. In this protocol we describe detailed methods for BEAMing, including a new technique for simultaneously generating 192 emulsions suitable for BEAMing.

  • ABIL WE09 (Degussa; see Supplementary Note online)

  • Binding buffer: 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl

  • Breaking buffer: 10 mM Tris-HCl (pH 7.5), 1% Triton-X 100, 1% SDS, 100 mM NaCl, 1 mM EDTA

  • Deoxynucleotide triphosphate (dNTP) mix (10 mM each; USB)

  • FACS Sheath Solution (BD Biosciences)

  • 5× hybridization buffer: 75 mM Tris-HCl (pH 9.5), 33.5 mM MgCl2, 25% formamide

  • Mineral oil (Sigma)

  • MyOne streptavidin-coated magnetic beads, C1, 1-μm diameter (10 mg/ml; 7–12 × 109 beads/ml; Invitrogen)

  • 0.1 M NaOH

  • 10× PCR buffer: 670 mM Tris-HCl (pH 8.8), 166 mM (NH4)2SO4, 100 mM β-mercaptoethanol, 11.7 mM MgCl2

  • Phusion Hot-start high-fidelity DNA polymerase, (2 U/μl; NEB)

  • Platinum Taq DNA polymerase (5 U/μl; Invitrogen)

  • Primers (see Fig. 1 and Box 1)

    Figure 1
    figure 1

    Outline of the BEAMing procedure.

    Full size image

  • Quant-iT PicoGreen dsDNA assay kit (Invitrogen)

  • Stainless steel beads (5 mm; Qiagen)

  • TE buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA

  • Tegosoft DEC (Degussa; see Supplementary Note)

  • TK buffer: 20 mM Tris-HCl (pH 8.4), 50 mM KCl

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Figure 1
Figure 2: Phase contrast micrograph (viewed at 400×) of emulsions deposited in the well of a 48-well tissue culture plate.
Figure 3: Analysis of bead populations with flow cytometry.

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Authors and Affiliations

  1. The Howard Hughes Medical Institute, and The Ludwig Center for Cancer Genetics and Therapeutics, The Johns Hopkins Kimmel Cancer Center, 1650 Orleans Street, Baltimore, 21231, Maryland, USA

    Frank Diehl, Meng Li, Yiping He, Kenneth W Kinzler, Bert Vogelstein & Devin Dressman

Authors
  1. Frank Diehl
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  2. Meng Li
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  3. Yiping He
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  4. Kenneth W Kinzler
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  5. Bert Vogelstein
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  6. Devin Dressman
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Corresponding author

Correspondence to Bert Vogelstein.

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Competing interests

Under a licensing agreement between EXACT Sciences and The Jonhs Hopkins University, K.W.K. and B.V. are entitled to a share of royalties received by the university on sales of products related to digital PCR. Under a licensing agreement between Agencourt Biosciences Corporation and The Jonhs Hopkins University, D.D., K.W.K. and B.V. are entitled to a share of royalties received by the university on sales of products related to the use of BEAMing for preparing templates for DNA sequencing. The terms of these arrangements are being managed by The Jonhs Hopkins University in accordance with its conflict of interest policies.

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Diehl, F., Li, M., He, Y. et al. BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions. Nat Methods 3, 551–559 (2006). https://doi.org/10.1038/nmeth898

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