Hot start PCR is a modified form of conventional polymerase chain reaction (PCR) that reduces the presence of undesired products and primer dimers due to non-specific DNA amplification at room (or colder) temperatures. Many variations and modifications of the PCR procedure have been developed in order to achieve higher yields; hot start PCR is one of them. Hot start PCR follows the same principles as the conventional PCR - in that it uses DNA polymerase to synthesise DNA from a single stranded template. However, it utilizes additional heating and separation methods, such as inactivating or inhibiting the binding of Taq polymerase and late addition of Taq polymerase, to increase product yield as well as provide a higher specificity and sensitivity. Non-specific binding and priming or formation of primer dimers are minimized by completing the reaction mix after denaturation. Some ways to complete reaction mixes at high temperatures involve modifications that block DNA polymerase activity in low temperatures, use of modified deoxyribonucleotide triphosphates (dNTPs), and the physical addition of one of the essential reagents after denaturation.

Through these additional methods, hot start PCR is able to decrease the amount of non-specific amplifications which naturally occur during lower temperatures – which remains a problem for conventional PCR. These modifications work overall to ensure that specific enzymes in solution will remain inactive or are inhibited until the optimal annealing temperature is reached. Inhibiting formation of non-specific PCR products, especially in early cycles, results in a substantial increase in sensitivity of amplification by PCR. This is of utmost importance in diagnostic applications of PCR or RT-PCR.

Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA segments by several orders of magnitude. The specific segments of DNA is amplified over three processes, denaturation, annealing and extension – where the DNA strands are separated by raising the temperature to the optimal from room temperature before primers bind and polymerase aligns nucleotides to the template strand. It uses DNA polymerase, which is slightly active at low temperatures. In conventional PCR, the reaction mix is completed at room temperature, and due to DNA polymerase activity, primers may form primer dimers or anneal to DNA non-specifically. During the PCR procedure, DNA polymerase will extend any piece of DNA with bound primers, generating target products but also nonspecific products which lower the yield. In hot start PCR, some of the reagents are kept separate until the mixture is heated to the specific annealing temperature. This reduces annealing time, which in turn reduces the likelihood of non-specific DNA extension and the influence of non-specific primer binding prior to denaturation.

In conventional PCR, lower temperatures below the optimal annealing temperature (50-65 °C) results in off target modifications such as non-specific amplifications where primers will bind non-specifically to the nucleic acid. These non-specific primer complexes, which are in excess in the mixture, are the cause behind the synthesis of by-products such as primer dimer and mis-priming. Mis-priming greatly impedes and reduces the efficiency of PCR amplification through actively competing with the target sequences for amplification. Similarly, primer dimers form complexes which decreases the amount of copy number amplifications obtained. This can be controlled by implementing hot start PCR which allows primer extensions to be blocked until the optimal temperatures are met.

In hot start PCR, important reagents (such as DNA polymerase and magnesium cofactors) are prevented from reacting in the PCR mixture until the optimal temperatures are met through physical separation or chemical modifications. Hot start PCR can also occur when the Taq polymerase is inhibited/inactivated or its addition is delayed until optimal annealing temperatures, through deoxyribonucleotide triphosphate modifications or by modifying the primers through caging and secondary structure manipulation.

Hot start PCR is often a better approach opposed to traditional PCR in circumstances where there is a low concentration of DNA in the reaction mix, the DNA template is highly complex, or if there are several pairs of oligonucleotide primers in the PCR.

Hot start PCR is a method which prevents DNA polymerase extension at lower temperature to prevent non-specific binding to minimise yield loss. Hot start PCR reduces the amount of non-specific binding through limiting reagents until the heating steps of PCR – limit the reaction early by limiting Taq DNA polymerase in a reaction. Non-specific binding often leads to primer dimers and mis-primed/false primed targets. These can be rectified through modified methods such as:

Enzyme linked antibodies/Taq DNA polymerase complexed with Anti Taq DNA polymerase antibodies: