from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

Melting curve analysis is a powerful and practical extension of real-time PCR. While real-time PCR focuses on collecting fluorescence at a single temperature each PCR cycle, melting analysis monitors fluorescence over time as the temperature is changing. Melting analysis fits nicely into the kinetic paradigm of PCR. Duplexes melt as the temperature increases, and the hybridization of both PCR products and probes can be monitored. Similar to "old" (slow) PCR being considered an equilibrium process, "old" (dot blot) hybridizations were performed at a single temperature. Dynamic monitoring of the entire melting curve as the temperature changes defines the entire melting transition, not just a single point (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Melting curve analysis was first integrated with real-time PCR on the LightCycler. No separations or reagent additions were required and melting analysis was fast (typically 2-15 min). The dye SYBR Green I conveniently provided quantification during PCR and melting analysis after PCR. The melting temperature of a DNA duplex is determined in large part by its sequence, G/C content and length. Specific PCR products can be easily distinguished from nonspecific PCR products. In many cases melting analysis eliminates the need for post-PCR processing such as gel electrophoresis. Genotyping by melting analysis was first demonstrated with a single hybridization probe and FRET to monitor probe melting. Different single base variants produced different probe stabilities, which were revealed by melting analysis. Later, dual hybridization probes were used for genotyping and both color and temperature multiplexing exploited. The use of a single fluorescein-labeled probe instead of two probes was a further simplification. Genotyping by melting without labeled probes was first shown with allele-specific PCR and SYBR Green I. Three primers were used, one with a GC-tail to discriminate alleles by melting temperature. Genotyping without GC-tails or labeled probes became possible with the availability of saturation dyes that detect heteroduplexes. These methods are detailed later in the section on high resolution melting analysis.