Practice question

Melting temperature, Tm, is the temperature at which half the primer-template duplex dissociates. Annealing temperature, TA, must be set below Tm to permit stable hydrogen bonding between primer and complementary template while maintaining specificity. Typically TA is adjusted 3-5°C below Tm, preventing non-specific binding caused by too low temperature and poor efficiency caused by temperature equal to or above Tm where duplexes are unstable. This empirical optimization balances yield and specificity and is calculated from GC and AT composition for each primer pair.