10/16/2014
Extreme PCR
Conflicts of Interest
Efficient Amplification in Less Than One Minute
• BioFire/Idaho Technology
Carl Wittwer, MD, PhD, Department of Pathology, University of Utah
– CoFounder and Chairman
• Royalties, Grants, Board
• Canon US Life Science
• Grants
• ARUP
• Grants
• NEB • Scientific Advisor
• Clinical Chemistry
• Associate Editor
23rd Annual Symposium on Molecular Pathology, Sept 16, 2014
Rapid‐Cycle PCR
(20‐60 second cycles)
How long does it take to….
• Denature
• Fast! (<1 sec)
• 30 cycles in 10-30 min
• Anneal
• Depends on [Primers], not [Product]
• Improved specificity
• Extend
• Complex
• Depends on [Polymerase]
BioTechniques cover: Jan. 1991
Melting curves after 30 cycles
Back to Water Baths….
20X [Primers] and [Polymerase]
30 sec Extreme PCR
Cold Bath
Control Capillary
Hot Bath
10 min Rapid Cycle PCR
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Cycling Speed is Determined by the Sample Container
Extreme vs Rapid Cycle PCR
5 µL sample volume
0.7 s/cycle
1.9 s/cycle
LC24
LightCycler
Extreme PCR compared to Rapid Cycle PCR
Real time Extreme Instrument
(45 bp human genomic target KCNE1)
Sample
Holder
Capillaries
NTC
Extreme PCR
(28 sec)
[Polymerase] (µM)30 sec PCR
[Primers] (µM)
1
10
NTC
Stepper
Motor
Optical
Stage
100 bp
50 bp
Rapid Cycle Primers
PCR
(12 min)
12 min PCR
0.064
0.5
HOT
WATER
COLD
WATER
Optics
Fiber
Sample Interrogation
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Polymerase and Primer Optimization IRL10RB (49 bp)
Polymerase and Primer Optimization NQO1 (102 bp)
58 sec PCR (30 cycles, 1.93 sec/cycle)
Extreme PCR Efficiency and Sensitivity
14.7 second PCR
91.7% (45 bp, 28 sec PCR)
60 bp AKAP10 (35 cycles, 0.42 sec/cycle)
95.8% (102 bp, 58 sec PCR)
Copies
40
15,000
1,500
150
15
1.5
NTC
Copies
15000
Fluorescence
Fluorescence
80
60
1500
150
15
1.5
NTC
20
15,000
1,500
150
15
1.5
NTC
60
40
0
10
20
30
40
0
50
15
1.5
NTC
10
20
30
40
50
Cycle Number
PCR Time
21.7 s
NTC
75 bp
50 bp
25 bp
8 µM polymerase, 20 µM Primers, 5 mM MgCl2
Polymerase and Primer Optimization
(Synthetic Template, 300 bp)
Quantification cycle
(Cq)
21.7 s
Quantification cycle
(Cq)
40
40
18.2 s
150
20
Cycle number
14.7 s
1500
0
0
11.2 s
15000
y = -3.538x + 39.231
R² = 0.9922
35
30
25
20
0
1
2
3
4
Log10(initial template copies)
y = -3.64x + 38.236
R² = 0.9909
35
30
25
20
0
1
2
3
4
Log10(initial template copies)
Minimum annealing/extension time dependence on product length
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Minimum extension time required for a 500 bp product
Extreme PCR for >100 bp Targets
(KAPA2G Fast polymerase)
KlenTaq1: One second required for every 60 bps
KAPA2G Fast: One second required for every 160 bps
Extreme PCR Challenges
• High [Polymerase] (20X)
•
•
Microfluidics for Extreme PCR and High Resolution Melting
(Canon U.S. Life Sciences)
Core Chip
Patent expirations (KlenTaq™)
Bulk sources (<$1/5 µl reaction)
• High [Primers] (20X)
• Hot start method
• Instrumentation
Inductive Heating / Forced Air Cooling
(BioFire Diagnostics/bioMerieux)
Continuous Flow PCR ‐ 45 bp genomic
(Thermal Gradient Device)
30 cycles in 45 seconds
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Is speed important?
30 min PCR
Extreme PCR Applications
• Infectious Disease • Critical Dx
• Epidemic Control/Triage
• BioThreat
• Point of Care/Impact
• Point of Consumption (Food/Water Testing)
• Intimate Encounters
• Drones
• Genetics
• Field Forensics
• In‐vitro fertilization
• Clinical Trials
• Oncology
• Intra‐operative
But….
• Who cares if you do PCR in 15 seconds if sample preparation takes 30 min?
• Molecular diagnostics requires:
• 1) sample preparation
• 2) amplification
• 3) analysis
Need for rapid sample preparation (<1 min)!
Summary
• Increase PCR speed: Jared Farrar
– 20X over rapid‐cycle PCR (10 min)
– 200X over regular PCR (2.5 hr) • Without sacrificing:
• Efficiency
• Sensitivity (single copy human genomic DNA)
• Specificity
• By altering chemistry:
• Increase [polymerase] 20X
• Increase [primers] by 20X
• Obtaining:
• 20X [product]
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PCR and Polymerase Activity
Jesse Montgomery
Definition of Polymerase Activity
The amount of polymerase required to incorporate 10 nanomoles
of radiolabelled dNTPs into activated salmon sperm DNA in 30 min
at 75 °C.
1. Montgomery JL, Rejali N, Wittwer CT. Anal Biochem. 2013 Oct 15;441(2):133‐9. • Template: “Activated” genomic DNA
2. Montgomery JL, Wittwer CT. Clin Chem. 2014 Feb;60(2):334‐40.
• Indicator: Radiolabeled dNTPs
3. Montgomery JL, Rejali N, Wittwer CT. J Mol Diagn. 2014 May;16(3):305‐13. • End‐point assay
• Buffer: Not standardized, not similar to PCR
Polymerase Assays
Polymerase Assays
(Linearity with Time)
(Linearity with Concentration)
Stopped‐flow Assay
Stopped Flow Assay
Polymerase Extension Rates
(nucleotides/sec/molecule)
Common Buffer
50 mM Tris, pH 8.3
500 ug/ml BSA
2 mM Mg++
Real‐time PCR Assay
Real‐time PCR Assay
Effect of PCR components on polymerase extension
Vendor Buffer
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Effect of monovalent cations
Effect of Tm Depressors
Effect of Dyes
Effect of incorporated bases
Effect of GC% and Temperature
Summary of PCR Component Effects
Probes
Probes (30‐40% activity)
Exo‐ faster than Exo+
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Thanks!
BioFire Dx / bioMerieux
NIH
ARUP
Roche Applied Science
Canon U.S. Life Sciences
State of Utah
University of Utah
http://dna.utah.edu
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