Extreme PCR Efficient Amplification in Less Than One Minute Carl Wittwer, MD, PhD, Department of Pathology, University of Utah Conflicts of Interest BioFire/Idaho Technology CoFounder and Chairman Royalties, Grants, Board Canon US Life Science Grants ARUP Grants NEB Scientific Advisor Clinical Chemistry Associate Editor 23 rd 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 Improved specificity Anneal Depends on [Primers], not [Product] Extend Complex Depends on [Polymerase] BioTechniques cover: Jan. 1991 Melting curves after 30 cycles 20X [Primers] and [Polymerase] Back to Water Baths. 30 sec Extreme PCR Cold Bath Control Capillary 10 min Rapid Cycle PCR Hot Bath 1
Cycling Speed is Determined by the Sample Container Extreme vs Rapid Cycle PCR 5 µl sample volume LC24 LightCycler 0.7 s/cycle 1.9 s/cycle Extreme PCR compared to Rapid Cycle PCR (45 bp human genomic target KCNE1) Extreme PCR (28 sec) Rapid Cycle Primers PCR (12 min) [Polymerase] (µm) 1 0.064 30 sec PCR 12 min PCR [Primers] (µm) 10 0.5 100 bp 50 bp Real time Extreme Instrument Capillaries HOT WATER Sample Holder Stepper Motor COLD WATER Optical Stage Optics Fiber Sample Interrogation 2
Polymerase and Primer Optimization IRL10RB (49 bp) Polymerase and Primer Optimization NQO1 (102 bp) 58 sec PCR (30 cycles, 1.93 sec/cycle) 14.7 second PCR 60 bp AKAP10 (35 cycles, 0.42 sec/cycle) Extreme PCR Efficiency and Sensitivity 91.7% (45 bp, 28 sec PCR) 95.8% (102 bp, 58 sec PCR) Fluorescence 60 40 20 Copies 15,000 1,500 150 15 1.5 15000 1500 150 15 1.5 Fluorescence 80 60 40 20 Copies 15,000 1,500 150 15 1.5 15000 1500 150 15 1.5 0 0 10 20 30 40 50 Cycle number 0 0 10 20 30 40 50 Cycle Number PCR Time 40 75 bp 50 bp 25 bp 11.2 s 14.7 s 18.2 s 21.7 s 21.7 s 8 µm polymerase, 20 µm Primers, 5 mm MgCl2 Quantification cycle (Cq) 40 y = -3.538x + 39.231 R² = 0.9922 35 30 25 20 0 1 2 3 4 Log 10(initial template copies) Quantification cycle (Cq) y = -3.64x + 38.236 35 R² = 0.9909 30 25 20 0 1 2 3 4 Log 10(initial template copies) Polymerase and Primer Optimization (Synthetic Template, 300 bp) Minimum annealing/extension time dependence on product length 3
Minimum extension time required for a 500 bp product (KAPA2G Fast polymerase) Extreme PCR for >100 bp Targets KlenTaq1: One second required for every 60 bps KAPA2G Fast: One second required for every 160 bps Extreme PCR Challenges High [Polymerase] (20X) Patent expirations (KlenTaq ) Bulk sources (<$1/5 µl reaction) Microfluidics for Extreme PCR and High Resolution Melting (Canon U.S. Life Sciences) Core Chip 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 4
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: 20X over rapid cycle PCR (10 min) 200X over regular PCR (2.5 hr) Jared Farrar Without sacrificing: Efficiency Sensitivity (single copy human genomic DNA) Specificity By altering chemistry: Increase [polymerase] 20X Increase [primers] by 20X Obtaining: 20X [product] 5
PCR and Polymerase Activity Definition of Polymerase Activity 1. Montgomery JL, Rejali N, Wittwer CT. Anal Biochem. 2013 Oct 15;441(2):133 9. 2. Montgomery JL, Wittwer CT. Clin Chem. 2014 Feb;60(2):334 40. 3. Montgomery JL, Rejali N, Wittwer CT. J Mol Diagn. 2014 May;16(3):305 13. Jesse Montgomery The amount of polymerase required to incorporate 10 nanomoles of radiolabelled dntps into activated salmon sperm DNA in 30 min at 75 C. Template: Activated genomic DNA Indicator: Radiolabeled dntps End point assay Buffer: Not standardized, not similar to PCR Polymerase Assays (Linearity with Time) Polymerase Assays (Linearity with Concentration) Stopped Flow Assay Real time PCR Assay Stopped flow Assay Real time PCR Assay Polymerase Extension Rates (nucleotides/sec/molecule) Common Buffer 50 mm Tris, ph 8.3 500 ug/ml BSA 2 mm Mg++ Vendor Buffer Effect of PCR components on polymerase extension 6
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+ 7
Thanks! BioFire Dx / biomerieux NIH ARUP Roche Applied Science Canon U.S. Life Sciences State of Utah University of Utah http://dna.utah.edu 8