How is Hot-Start Technology Beneficial For Your PCR?

Nonspecific amplification is one of the major issues that can drastically impact PCR performance resulting in one or more of the following outcomes:

  • Low yield of target amplicon
  • Loss in sensitivity of target amplicon
  • Poor interpretation of results
  • Poor efficacy in downstream applications.

A common source of nonspecific amplification is the extension of misprimed targets by DNA polymerases and the formation of primer-dimers. One workaround that researchers use to avoid nonspecific amplification is to prepare the PCR reaction mixture on ice. The reduced temperature helps keep the activity of the DNA polymerase low, however synthesis of undesirable products may still occur before the start of PCR. Another solution is to use a hot-start DNA polymerase.  Hot-start technology and the associated modifications inhibit DNA polymerase activity at room temperature effectively preventing spurious nonspecific amplification. 

How does hot-start technology work?

Methods of hot-start PCR employ an enzyme modifier such as an antibody, affibody or aptamer. Two of the most common methods used are chemical modification and antibodies. While they both inhibit polymerase activity at room temperature, there are some key differences between the two. 

Hot-start technology Advantages Disadvantages

Polymerases are chemically modified to block their activity at room temperature

  • Hot-start modification permits more stringent conditions resulting in increased specificity
  • Free of animal origin components
  • Require longer re-activation times in order for the polymerase to become fully active
  • Chemical modifications can affect polymerase performance for amplicon lengths of 3kb or more

Antibodies specific to the polymerase are used to block activity of the polymerase at room temperature

  • Antibodies have no effect on polymerase performance, so all enzyme features are intact as in the non-hot-start enzyme
  • High specificity
  • No additional time required as the initial denaturation step during PCR cycling is sufficient to activate the polymerase
  • Antibodies can be of animal origin

What are the benefits of hot-start technology?

  • Prevents primers binding to template sequences with low homology (mispriming).
  • Prevents primers binding to each other (primer-dimer formation) during reaction setup, prior to the start of PCR.
  • Increases sensitivity and yield of the desired target fragment.
  • Hot-start polymerases enable reaction setup at room temperature without sacrificing specificity making it easier to run a large number of reactions or to use automated liquid handling platforms for PCR.

Where can I learn more?