Extreme PCR: Robust, Efficient Amplification in < 1 Min
Conference Recording Jul 14, 2014
About the Speaker
Carl Wittwer, MD, PhD is a professor of Pathology at the University of Utah. He introduced rapid cycle PCR in the early 1990s and was the primary inventor of the LightCycler system for real time PCR. In the 2000s, he also developed high resolution melting (HRM) for genotyping and variant scanning. He is currently chairman of BioFire Diagnostics (formerly Idaho Technology), still focused on increasing the speed and performance of targeted DNA diagnosticsAbstract
Rapid-cycle PCR was introduced in 1990, reducing 2-4 hours of amplification to 10-15 min. Since that time, incremental progress has been made in reducing amplification times below 10 minutes, although yield and PCR efficiency often suffer at higher speeds. We now introduce, “Extreme PCR”, a method that combines <2 s thermal cycles with chemistry modifications to enhance the completion of annealing and extension segments. Specifically, primer and polymersase concentrations are increased so that primer annealing and complete extension near 100% efficiency each cycle. Cycle times of less than 1 s were obtained for PCR products less than 100 bp, resulting in specific, high-yield amplification as demonstrated by gels and melting curves. In addition, we introduce a new method for measuring polymerase activity under PCR conditions. Performed on real-time instruments, the effect of various components on extension rates reveal that monovalent cations, most Tm depressants, secondary structure, probes and dyes inhibit extension, while Mg++, temperature, GC content, and DMSO have local maxima. By studying the effect of different PCR components on extension rate, amplification speed can be optimized.