by K Chico, Jessica Piczon

Polymerase chain reaction (PCR) is a versatile nucleic acid amplification process used across many fields, including molecular biology and forensics. Traditional PCR setups employ a mixture of two specific oligonucleotide primers, a thermostable polymerase, dNTPs, a magnesium-containing reaction buffer, and template sources from either DNA or RNA.

One common issue related to unreliable PCR performance is the incidence of off-target amplifications that result from nonspecific primer binding and extension. These issues are thought to occur during the cooler, less stringent, thermal cycling conditions at which many polymerases are still partially active.

A number of techniques, collectively termed “hot start” PCR approaches, have been devised to reduce these off-target amplifications; the goal is to block polymerase extension at lower temperatures, until the primer annealing temperature is reached. For some applications, hot start PCR may be the only way to successfully amplify the desired products while maintaining a high yield and excellent specificity.  

Preliminary hot start PCR approaches were achieved by withholding a key component during reaction setup, as in polymerase, dNTPs, primers, or Mg2+, and then introducing them once the reaction mixture reached a desired, elevated, temperature. These methods use physical, solid, barriers like wax pellets to segregate reaction components until the hot start activation temperature is achieved. At this step, the wax melts and the amplification reaction proceeds as normal, providing for a substantially easier and safer, uninterrupted, PCR process.

Over time, these barriers have been simplified through the creation of pre-molded wax beads that encapsulated reagents which are then released at a certain temperature. More recent developments include the use of microfluidic devices to precisely control or inhibit the addition of components into a PCR reaction.

Another hot start approach is to alter polymerase to effectively block molecular activity until hot start activation. This has been performed through the use of antibodies, aptamers, chemical modifications, protein fusions, temperature-sensitive amino acid point mutants, and temperature dependent ligands which initially bind to polymerase prior to amplification. In this method, the inhibitory molecule is selectively denatured once a certain reaction temperature is reached. After, the fully active polymerase can effectively initiate the polymerization reaction.

The most recent development in hot start PCR approaches incorporates the use of thermolabile chemical modifications to oligonucleotide primers and dNTPs. One notable modified primer construct includes those that have the ability to improve hybridization selectivity. Other primer modifications include those that are removable by 3'-5' exonuclease activity, UV irradiation, or even thermal deprotection. Similarly, dNTPs have been modified with thermolabile groups, which are subsequently removed once a certain reaction temperature is reached.