Rapid Cycle and Extreme Polymerase Chain Reaction

Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for speed; sensitivity, specificity, and yield are equivalent or better than conventional PCR. What is required (and not widely available) is rapid, accurate control of reaction temperature during cycling. Specificity improves with cycling speed, and efficiency can be maintained by increasing polymerase and primer concentrations. Speed is aided by simplicity, dyes that stain double-stranded DNA are less expensive than probes, and one of the simplest polymerases, the deletion mutant KlenTaq, is used throughout. Rapid amplification can be coupled with endpoint melting analysis to verify product identity. Instead of commercial master mixes, detailed formulations for reagents and master mixes compatible with rapid cycle and extreme PCR are described.