Emulsion PCR One the basic technique of NGS

1. Emulsion Polymerase Chain
Reaction
Presented by:
Salman Jamil
(2014-bte-012)
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2. What is Emulsion PCR?
“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.”
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3. Principle
The basic principle of ePCR is dilution and
compartmentalization of template molecules in water
droplets in a water-in-oil emulsion.
Each droplet contains a single template molecule and
functions as a micro-PCR reactor.
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4. Procedure
Fragmentation of DNA
• The sample is fragmented ranging from 300 to
800bp.
Ligation of adapters
• Adapters with one end sticky and one blunt are ligated
with fragments.
• Phosphates are removed from sticky ends to avoid
dimerization.
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5. Procedure
 Formation of clonal bead populations
• Beads coated with
streptavidin are used.
• Beads have primers that
matches the adapters used.
• Each bead is emulsified in a water-in-oil droplet with
PCR reagents (DNA polymerase, primers, buffers,
dNTPs).
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6. Formation of clonal bead
populations
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7. Amplification
Denaturation to single strands
• The double stranded DNA's with adapters are
then denatured by heating the DNA up to 95 °C.
 Annealing of ssDNA
• The ssDNA is then attached to the beads.
• Reverse strand (bottom strand or 3’-5’ strand) anneal
to the f-primer on bead surface and r-primer anneal
to the forward strand (top strand or 5’-3’ strand)
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8. Amplification
Extension
• Polymerase amplifies the forward strand (5’-3’)
starting from beads towards the primer site.
• The reverse strand is amplified starting from primer
towards the bead site.
 Cycle
• Each newly formed 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.
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10. Procedure
Emulsion Breaking
• After amplification, the emulsions are broken using
isopropanol and detergent buffer. The solution is
then vortexed, centrifuged, and magnetically
separated.
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11. Thanks!
Its end now.
I hope you learn
something new
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