There are a number of factors that can affect the specificity of the amplification reaction. The stringency of the annealing step can be controlled to some extent by adjusting the annealing temperature. Minimising the incubation time during the annealing and extension steps will limit the opportunities for mispriming and extension by molecules of otherwise idle DNA polymerase. Reducing primer and enzyme concentrations also serves to limit mispriming, particularly the type that leads to dimerisation. Finally, changing MgCl2 levels can further improve specificity, either by increasing the stringency of the reaction or by direct effects on the polymerase itself (activity, processitivity, etc.).
© 2001 Alexander Binder