Hot start PCR is the modification of the conventional PCR which reduces the non-specific bindings by limiting one of the reagents until the heating step of the PCR. Conventional PCR often results in the amplification of non-target sequences that are also present in the reaction mix. Hot start PCR allows for reaction set up at room temperature without non-specific amplification and primer dimer formation. The yield and accuracy of the results can also be increased. Hot start PCR is designed to inhibit Hot Start Taq DNA polymerase activity, or the incorporation modified dNTPs during reaction set up until a heat activation step occurs. Various methods are available to halt hot start polymerase activity, including chemical modification, antibody-mediated, and aptamer-mediated technology.
Fig. 1 Comparison of conventional PCR and hot start PCR
The hot start PCR technique could decrease the nonspecific bindings. Also, it prevents mis-priming and formation of primer dimer. By using the hot start Taq DNA polymerase, the reaction can even be prepared at room temperature. More importantly, it increases the yield and accuracy of the results. However, hot start PCR has some limitations. The cost of this technique is relatively high because of the use of the antibody and the wax beads. Another disadvantage is that it can not amplify the DNA templates that larger than 2kb. The heating step is very important in the hot start PCR, and the template DNA can be damaged or break down seriously because of the higher temperature for a longer time.
Initially, hot start PCR was performed by limiting the concentration of Mg2+, dNTP or enzyme. Alternatively, hot start can be realized by separating the reaction components with a wax bead barrier layer that melted when the mixture is heated during the initial denaturation step of the PCR. In either case, when nonspecific binding may occur between primers and nonspecific DNA targets, the hot start will prevent the new DNA from aggregating at the initial stage of the reaction. Recently, chemically or antibody engineered polymerases have been commercially available, and once they reach a certain temperature, they are activated. Some commonly used methods are as follows:
(1) Removal of Essential Reaction Components
The first attempt at hot start PCR simply delayed the addition of PCR until the reaction temperature reached 94 ℃ at the time of DNA complete denaturation. This method is feasible, but additional processing steps are required, which is not convenient when performing multiple reactions and the sample is more likely to be contaminated.
(2) Sequestration of Components within the Reaction
Most of the methods here use heat sensitive materials, such as wax or agarose beads, to separate a necessary component (e.g., Taq DNA polymerase or MgCl₂) from the rest of the reaction mix. The sequestered component is released into the reaction mix once the reaction temperature is high enough to melt the wax or agarose, allowing PCR to be carried out. Another method of chelation relates to the formation of magnesium deposits. The precipitate chelates magnesium ions from the reaction mixture (required by the polymerase) and releases them at a reaction temperature between 50 and 95 ℃.
(3) Chemical Modification of the Polymerase
In the hot start PCR method, the polymerase is reversibly inactivated by chemical modification. Reactivate the enzyme by preheating the reaction mixture at 94 - 95 ℃ for 9 - 12 minutes. This may be problematic because high temperature reactivation can lead to the purification of DNA templates, thereby reducing the quality of the products produced. In addition, the long reactivation time increased the PCR reaction time and decreased the throughput.
(4) Antibody Modification of the Polymerase
Antibodies specific to polymerases are added to block the polymerase activity at room temperature. In the denaturation step, the antibody releases the enzyme and is denatured. The free enzyme is usually active and the reaction happens. Antibodies come from animals, and sometimes pollutants interfere with PCR. Unlike chemically modified polymerases, no additional time is required in the initial denaturation step.
Fig. 2 TaqStart Antibody inhibits polymerase activity before thermal cycling begins. TaqStart is an anti-Taq monoclonal antibody that inhibits the enzymatic activity of Taq and Taq-derived DNA polymerases during room temperature reaction assembly TaqStart Antibody with Taq-derived DNA Polymerase is added during PCR set-up.
(5) Ligand-mediated Polymerase Inhibition
The most promising hot start methods use oligonucleotides or antibody ligands to reversibly inhibit polymerase. Ligand-mediated inhibition is superior to inactivate the polymerase by chemical modification because a lengthy, high temperature reactivation step is not needed. When the ligands were simply dissociated or denatured at high temperature, the enzyme activity was restored. Once the inhibition disappears, the polymerase will retain most of its original activity.