Real-Time Polymerase Chain Reaction and Melting Curve Analysis

Monitoring polymerase chain reaction (PCR) once each cycle is a powerful method to detect and quantify the presence of nucleic acid sequences and has become known as "real-time" PCR. Absolute quantification of initial template copy number can be obtained, although quantification relative to a control sample or second sequence is often adequate. Melting analysis following PCR monitors duplex hybridization as the temperature is changed and is a simple method for sequence verification and genotyping. Melting analysis is often conveniently performed immediately after PCR in the same reaction tube. The fluorescence of either DNA dyes that are specific to double-strands or fluorescently labeled oligonucleotide probes can be monitored for both real-time quantification and melting analysis. When used together with rapid temperature control, these methods allow amplification and genotyping in less than a half hour.