How does HotStart PCR help minimize nonspecific amplification events?
HotStart PCR is a technique commonly used to improve the sensitivity and specificity of PCR amplifications. Lack of sensitivity or specificity is most often caused by the amplification of nonspecific priming events, such as primer dimers, that usually occur at the lower temperatures when reactions are set up. Although thermostable DNA-dependent DNA polymerases have optimal activity at higher temperatures, they do also have some activity at lower temperatures when they may amplify these nonspecific priming events. HotStart enzymes are inactive at room temperature, and require heating at nucleic acid melting temperatures in order to be activated. In this way, nonspecific priming events are melted before the enzyme can amplify them. During PCR cycles, the temperature never drops low enough during annealing of gene-specific primers for nonspecific priming events to occur, resulting in amplification exclusively of the target of interest. When using a HotStart DNA polymerase, it is critical that the initial denaturation step in the experiment be of sufficient duration to fully activate the enzyme.