### Melting temperature

The melting temperature is the temperature at which 50% of the primer concentration are found in the form of the primer-template duplex. Hence, the melting temperature indicates the stability of primer-template duplexes. High melting temperatures indicate energetically favorable primer-template interactions, while low melting temperatures indicate low affinities. Melting temperature is of concern for PCR for several reasons:

- Primers that are to be used together in the same PCR run should have similar melting temperatures such that each primer can operate close to its optimal annealing temperature, thereby maximizing product yield and quality.
- Primers with large melting temperatures require high annealing temperatures, which lead to fewer primer-template hybridization events and reduced product yields.
- Primers with low melting temperatures require low annealing temperatures. At low annealing temperatures, primer hybridization can be unspecific, leading to undesired PCR products, which are caused by primers binding with mismatches.

Typical melting temperatures lie between 55 and 65 degrees Celsius. openPrimeR uses MELTING for the computation of melting temperatures.