Several methods exist for performing hot start including manual techniques, using barriers and reversible polymerase inactivation. Examples of unwanted reactions which can occur at lower temperatures prior to PCR amplification. Manual hot start methods require the researcher to withhold a critical component, usually magnesium or the polymerase, until the reaction has been heated. The withheld component then is added to initiate the reaction. This method requires extra manipulations of the sample and increases the risk of contamination.
Most barrier methods use wax and require a two-step approach.
The wax bead is melted and allowed to cool, forming a barrier of solidified wax over the aqueous phase. The critical component is then added to the surface of the solidified wax layer. Upon reheating, the wax barrier melts, allowing the final component to mix with the pre-added components in the aqueous phase. Finally, a third method of hot start amplification is reversible polymerase inactivation. Upon heating, the compound dissociates from the polymerase, restoring enzyme activity.