Jared S Farrar1, Carl T Wittwer2
1Virginia Commonwealth University;
2University of Utah
BACKGROUND: PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown.
METHODS: We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62°C–76°C and denaturation temperatures of 85°C–92°C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring.
RESULTS: Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles.
CONCLUSIONS: The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.
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