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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. Non-specific binding is 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.

Background
Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA segments by several orders of magnitude. 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.

Hot start PCR is especially useful in place of conventional PCR if the reaction mix contains little DNA, if there is expected non-specific binding in a multiplex PCR reaction , and if the expected amplicon sizes are very small (less than 150bp).

Methods
The general principle of hot start PCR is the isolation of one or more reagents from the reaction mix, until the mixture is heated up to denaturation temperature.

Physical Separation
Early methods of hot start PCR involved excluding or limiting the concentration of one of the reagents, until the denaturation stage begins. Over time, a wax-isolation technique was developed whereby a wax layer isolated the reagent until the initial stage of PCR, when it dissolved and released the isolated reagent.

DNA polymerase inactivation
DNA polymerase inactivation is a method where the DNA polymerase is kept inactive until the PCR mix is prepared. This can be done using antibodies, and aptamers (chemical antibodies), affibodies (smaller antibody-mimics targetting peptides), as well as chemical modifications. Antibody/affibody/aptamer inactivation of DNA polymerase is more suited for situations requiring a fast inactivation time. When the inactivation of DNA polymerase is not time-sensitive, chemical modifications can be used.

Deoxyribonucleotide triphosphate (dNTP) modifications
Chemically modified deoxyribonucleotide triphosphates (dNTPs ) start binding to the template DNA only after the reaction mix is heated up.

Advantages
Hot start PCR significantly reduces nonspecific priming, formation of primer-dimers, and often increases product yields. It also requires less handling and reduces the risk of contamination.

Disadvantages
If the DNA polymerase is chemically modified, the re-activation time during the initial denaturation stage of the PCR cycle is increased because there is a waiting period while the enzyme activates. This increased heating time can damage DNA. Additionally, studies have shown that some chemical modifications can cause issues in amplifying long strands of DNA.