02. Emulsion PCR ePCR

Emulsion PCR is a PCR variation that some NGS technologies use to replicate DNA sequences. It is conducted on a bead surface within tiny water bubbles floating on an oil solution.

Why replicate DNA?

This is a very important concept to understand, as all NGS techniques replicate DNA before sequencing is done. In short, DNA is replicated in order to amplify signals. No matter the method of sequencing, without a proper amount of amplification, it's near impossible to detect each base call.

Procedure

1) Fragmentation of DNA

The library is first fragmented either by sonication (high sound energy) or nebulization (forces DNA through a small hole) to fragments ranging from 300 to 800 bp.

2) Adapters are ligated

Adapters are then ligated onto the DNA fragments. These allow the strands to bind to the emulsion beads.

3) Denature to single strands

The double stranded DNA's with adapters are then denatured by heating the DNA up to 95 °C. Denaturing DNA simply means to go from double stranded DNA (dsDNA) to two single strands (ssDNA) - the hydrogen bonds keeping the two together are broken.

4) Formation of clonal bead populations

Each bead coated with streptavidin, which is resistant to organic solvents, denaturants, detergents, proteolytic enzymes and extremes of temperature and pH.

Over a billion beads are used with a primer that matches the adapters attached earlier. The ssDNA is then attached to these beads.

Each bead is emulsified in a water-in-oil droplet with PCR reagents (DNA polymerase, primers, buffers, dNTPs).

Emulsion PCR components
Emulsion PCR components.

5) ePCR amplifies DNA strands on beads

Within these droplets, PCR is conducted. This involves the steps Denaturation, Annealing, Elongation. Firstly, the strand is elongated with DNA polymerase and dNTPs. Then the double-strand is denatured, allowing for the strand to ligate to another site on the surface of the bead. Eventually, 1 million copies of the target is amplified on the surface of each bead. The water-in-oil droplet is approximately 1-um.

Follow the figure to see how each bead is able to replicate DNA on its surface.

ePCR amplifies DNA strands on beads.
ePCR amplifies DNA strands on beads. Figure adapted from Andy Vierstraete.

6) Emulsion Breaking

After the DNA strands are amplified, the emulsion from the preceding step is broken using isopropanol and detergent buffer. The solution is then vortexed, centrigued, and magnetically separated. The resulting solution is a suspension of empty, clonal and non-clonal beads, which will be filtered in the next step.

7) Bead enrichment

After PCR is conducted, you are left with a mixture of some beads that have amplified DNA attached on its surface, and some that do not.

We may take out the enriched beads by attaching streptavidin coated magnetic enrichment bead. With a magnet, we can then pull out the beads with amplified DNA.

Emulsion PCR components
Bead enrichment using magnets.

There are other methods of bead enrichment that include using larger beads that are able to bind to beads with amplified DNA. After centrifugation, the beads with amplified DNA and without can then be separated.

8) Bead Capping

Attach a capping oligonucleotide to the 3' end of both unextended forward ePCR primers and the RDV segment of template DNA. This helps in coverslip arraying, which is used to polony sequencing, and prevents fluorescent probes from ligating to the ends.

9) Result

The beads with amplified sequences are then placed on a slide and are sequenced. Due to their high density of the same DNA molecule, the signal is amplified, allowing computers to read the sequencing data.

References

  1. Emulsion PCR figure adapted and used with permission from Andy Vierstraete.

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