from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

PCR specificity under ideal conditions is extraordinary. However, the genomes are large and primers may bind not only to their intended target but also to other areas of the genome. Furthermore, the primers in PCR are at high concentrations, so even minor self or cross complementation may initiate primer dimers. These side reactions can create so-called "non-specific" products other than the desired product. A number of methods have been developed to prevent primer extension at low (room) temperatures where polymerase activity, although greatly reduced, is still capable of extending primers (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

The first "hot-start" techniques relied on adding an essential reaction component (such as the polymerase) only after the reaction had reached high temperatures to favor specific primer annealing. This required opening the reaction container and increased the possibility of PCR contamination. To circumvent this problem, waxes and greases were used to physically partition reagents with a barrier that would melt at high temperature, mixing the essential reagent(s) with the other reaction components (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Instead of physical separation, polymerase activity can be inhibited at room temperature. For example, monoclonal antibodies against the active site of the polymerase can inhibit the enzyme until they denature at high temperature. Alternatively, the polymerase active site can be chemically modified by heat-labile covalent modifications that break down and activate the enzyme at high temperature. Instead of inactivating the polymerase, oligonucleotide primers or dNTPs can be modified at their 3'-end with similar heat-labile linkages (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Many different reagents are now available to augment PCR specificity, but they are usually only necessary when the template copy number is low. Nevertheless, such reagents are an easy way to increase the robustness of PCR, sometimes making optimization unnecessary. If a hot start method is required, the best method depends on the circumstances. For example, an antibody-mediated hot-start is more useful in rapid PCR because chemically modified polymerases typically require 15-30 min for activation, longer than an entire rapid-cycle PCR protocol.