Emulsion PCR

For Ion Torrent PGM, but basic ideas apply to other platforms

BINF 6150 Fall 2015

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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

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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

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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-stranded2. Anneal - cool, primers anneal to template DNA3. Extend - heat, polymerase makes DNA using template

• At end of each cycle, twice as much template

Double-stranded library molecule

• P' anneals to oligos on beads for emulsion PCR • A anneals to sequencing primer - sequencing by synthesis

5

3'AP'

P3' 5'

A'

5'

top strand

bottom strand

unknown sequence

bar code

Bead with single-stranded oligos

• ISP - ion sphere particle (not to scale)• coated with P1 oligonucleotides - millions

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ISP

P'

P'

P'

P'

P'

P'

3'

3'

3'

3'

3'

3'

attachment site

Contents of single micelle (reactor)

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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

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

Emulsion PCR - 1st Anneal

• Primer P' (on bead) anneals to bottom strand• Primer A anneals to top strand

3'

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3'AP'

P'

3'

5' 3'5'

A'

5'

A'P

5'

bottom strand

top strand

ISP

Emulsion PCR - 1st Extend

• DNA polymerase synthesizes new DNA• Extends 3' end of P', A primers

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3'AP'

P'

A'P

5'3'

5' 3'

5'

A'

5'3'

ISP

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'

AISP

Emulsion PCR - 2nd Denature

• Strands separate 12

3'AP'

P'

A'P

5'3'

5'

5'5'

3'

3'A

ISP

Emulsion PCR - 2nd anneal

• P' primer anneals to bottom strand• A' primer anneals to top strand

AP'

13

3'

P

5'3'5'

5'3'

P'5' 3'

A'ISP

5'5'

5'

3'

3' 5'

P'

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

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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

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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:

220 = 1,048,576

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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