BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions (2024)

The most important biotechnological advances made in the 20^th 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)

  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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