“Hot Start” PCR using VERSA™ Mini PCR Setup Workstation

PCR is a highly sensitive technique, enabling researchers to rapidly select and amplify a specific target sequence from a background of non-related sequences. However, if all reaction components are combined at the same time, at room temperature, it is possible that non-specific annealing will occur between fragments. Such premature mis-priming events may amplify non-specific DNA sequences - especially if target sequences exist in low copy numbers. Time, money, and resources are wasted when PCR tests are redone to check questionable results.

One solution is “hot start” PCR. This method requires one or more essential reaction components to be excluded until the reaction temperature exceeds the annealing temperature of the target primers. One variation of this procedure uses paraffin wax beads embedded with the omitted PCR reagents. Beads are manually added to PCR plates, followed by the remainder of the reagents prior to thermocycling. The wax melts once exposed to sufficient heat, releasing the trapped reagents and enabling the reaction to proceed normally. Cooling the plate causes the paraffin to rise to the surface and solidify, trapping the reaction liquid beneath a wax cap. The cap must then be pierced to allow removal of the sample.