Hot start PCR (hot start PCR) refers to PCR in which Taq DNA polymerase only functions when the sample temperature exceeds at least 70°C, which improves the specificity of the reaction. Taq DNA polymerases are generally more active at temperatures much lower than optimum. During the initial heating of the PCR reaction, before the sample temperature rises to 70°C, the primers may form non-specific binding to part of the single-stranded template at lower temperatures and extend under the action of Taq DNA polymerase. As a result, non-target sequences can be amplified, affecting the specificity of the reaction. Hot start can reduce the amplification of non-target sequences and improve the specificity of the reaction. Hot-start PCR is particularly useful when primer design is limited by the location of genetic elements, such as site-directed mutations, expression cloning, or the construction and manipulation of genetic elements for DNA engineering.

The basic method is: before the reaction, do not add Taq DNA polymerase, dNTPs and primers to the sample tube, but heat the sample. When the temperature rises above 70°C, add the above reagents to start the PCR reaction. .

A hot start delays DNA synthesis by inhibiting an essential component until the thermal cycler reaches the denaturing temperature. Manual hot-start methods that delay the addition of Taq DNA polymerase are cumbersome, especially for high-throughput applications. Other hot-start methods use a wax shield to isolate one of the essential components (magnesium ions, enzymes, templates, buffers, etc.). During thermal cycling, the wax melts and releases the various components and mixes them together. The wax shield method is also cumbersome, prone to contamination, and not suitable for high-throughput applications.