Describes using emulsion PCR to decorate Ion Sphere Particles (beads) with library molecules for Ion Torrent PGM sequencing. This is from a class Ann taught on Genomic Biotechnology.

1. Emulsion PCR
For Ion Torrent PGM, but basic ideas
apply to other platforms
BINF 6150 Fall 2015
1

2. Emulsion PCR - emPCR
• Emulsion - mixture of insoluble liquids
– example) oil and water, salad dressing
• Emulsify (mix) oil and water makes emulsion
– microscopic oil droplets in water
– droplets called micelles
• Oil droplets are like plastic tubes in ordinary PCR
– Each droplet has different template
2

3. Emulsion PCR - for sequencing
• Emulsify solution w/ library, nucleotides, buffer,
primers, taq polymerase, Ion torrent bead (ISP -
Ion Sphere particle), oil
• Aim for: 1 library molecule per droplet
– This is why need to quantify library
• At end, get beads coated with many copies of one
library molecule only
3

4. 4
Reminder: PCR
• Contains DNA template (dsDNA), primers, heat-stable
DNA polymerase, nucleotides, buffer
– primers in excess (otherwise template re-anneal)
• Repeat three steps - called "cycle"
1. Denature - heat, dsDNA becomes single-stranded
2. Anneal - cool, primers anneal to template DNA
3. Extend - heat, polymerase makes DNA using template
• At end of each cycle, twice as much template

5. Double-stranded library molecule
• P' anneals to oligos on beads for emulsion PCR
• A anneals to sequencing primer - sequencing by synthesis
5
3'
AP'
P
3' 5'
A'
5'
top strand
bottom
strand
unknown
sequence
bar
code

6. Bead with single-stranded oligos
• ISP - ion sphere particle (not to scale)
• coated with P1 oligonucleotides - millions
6
ISP
P'
P'
P'
P'
P'
P'
3'
3'
3'
3'
3'
3'
attachment site

7. Contents of single micelle (reactor)
7
3'
only one
AP'
P'
A'
P
5'
3'
5' 3'
5'
A'
5'
top strand
bottom
strand
ISP
in excess
also, nucleotides, buffer, polymerase

8. Emulsion PCR - 1st denature
• Top and bottom strands separate 8
3'
AP'
P' A'
P
5'
3'
5' 3'
5'
A'
5'
top strand
bottom
strand
ISP

9. Emulsion PCR - 1st Anneal
• Primer P' (on bead) anneals to bottom strand
• Primer A anneals to top strand
3'
9
3'
AP'
P'
3'
5' 3'
5'
A'
5'
A'P
5'
bottom
strand
top strand
ISP

10. Emulsion PCR - 1st Extend
• DNA polymerase synthesizes new DNA
• Extends 3' end of P', A primers
10
3'
AP'
P'
A'P
5'3'
5' 3'
5'
A'
5'
3'
ISP

11. Emulsion PCR - end of 1st extend
• Two copies of library molecule
• New top strand attached to bead 11
3'
AP'
P'
A'P
5'3'
5'
5'
A'
5'
3'
3'
A
ISP

12. Emulsion PCR - 2nd Denature
• Strands
separate
12
3'
AP'
P'
A'P
5'3'
5'
5'
5'
3'
3'
A
ISP

13. Emulsion PCR - 2nd anneal
• P' primer anneals to bottom strand
• A' primer anneals to top strand
A
P'
13
3'
P
5'3'
5'
5'3'
P'
5' 3'
A'
ISP
5'
5'
5'
3'
3' 5'
P'

14. 14
3'
AP'
A'P
5'3'
5'
5'3'
P'
5' 3'
A
ISP
5'
5'
5'
5'
3'
3'
3'
3'
Emulsion PCR - 2nd extend

15. 15
3'
AP'
A'P
5'3'
5'
5'3'
P'
5' 3'
A
ISP
5'
5'
5'
5'
Emulsion PCR - end of 2nd extend
• 4 copies
library
molecule
• 3 copies
top
strand
attached
to bead

16. And repeat
• Number of molecules doubles each cycle
• After each cycle
– twice as many P primers extended
– twice as many templates for further extension
• To illustrate, # of library molecules after 20 cycles:
16
220 = 1,048,576

17. Summary
• Emulsion PCR happens in reaction chamber called
a "micelle"
• Need 1 library molecule per micelle
• Would like to have one bead per micelle, but
probably OK if more than one
– depends on application
• Next: Once you decorate beads, then what?
– ES enrichment
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