Abstract

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 <2s 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 1s 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.