How does HotStart PCR help minimize non-specific 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 non-specific priming events, such as primer dimers, that usually occur at the lower temperatures where reactions are set up. Although the thermostable DNA-dependent DNA polymerases have optimal activity at higher temperatures, they do also have some activity at lower temperature where they may amplify these non-specific 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, the non-specific priming events are melted before the enzyme can amplify them. During the PCR cycles, the temperature never drops low enough during the annealing of the gene-specific primers for the non-specific priming events to re-occur, resulting exclusively in amplification of the target of interest. When using a HotStart
