Why should I use a Hot Start polymerase?

The main reason to us a hot start Polymerase (used within a process also known as a ‘Hot Start PCR’) in your PCR reaction is to avoid unspecific amplification. Most DNA polymerases work best at a temperature between 68 and 72°C. In some cases, an enzyme can become slightly active below these temperatures and this will cause unspecific binding, leading to unspecific amplification. A hot start PCR will reduce the nonspecific amplification significantly.

What is a Hot Start polymerase? Technically it is a standard PCR polymerase, but a Hot Start Polymerase is inhibited in its functionality by a structural change to the enzyme. These changes can include antibody interaction, aptamer technology or chemical modification. Typically a Hot Start polymerase needs to be activated by incubating the enzyme at 95°C for a longer period of time to remove the polymerase inhibitor.

Last but not least, when using a hot start polymerase, it provides the advantage of performing the PCR reaction setup at room temperature. Usage of a Hot Start polymerase can therefore be advised when performing high-throughput experiments using liquid handlers or experiments demanding high specificity.

So why not always use a hot start polymerase? Well, it has a disadvantage as well.. The re-activation time during the denaturation stage is increased for activation of the enzyme. This increased heating time could damage your DNA. Studies have also shown that using a hot start PCR can cause issues when amplifying long strands of DNA.

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