Why would running touchdown denaturation cycles increase specificity of a PCR?
Sometimes you will need to run a touchdown cycle for the annealing step to decrease non-specific binding of primers. But I would like to know why slowly decreasing the temperature of denaturation step would also increase specificity.
I already know about the annealing temperature. The reason why I asked this is because I received a touchdown DENATURATION protocol for a certain primer and it worked after countless attempts to fiddle with the annealing temperature.
- 1 decade agoFavorite Answer
The principle behind a touchdown protocol is to give the most specific product a head start. You don't slowly decrease the temperature of denaturation (that remains fixed at ~95), Instead you decrease the annealing temperature.
Templates that anneal perfectly to the primers will have a higher Tm & be the first to start amplifying. Templates which only partially match the template or each other (primer dimer) won’t start amplifying until the annealing temperature drops in later cycles.
Usually the concern is primer dimer or repeat sequences in the template because shorter products can out compete longer products during cycling.Source(s): frequently use touchdown protocols