Hot start PCR

Hot start PCR is a modified form of polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA by inactivating the DNA polymerase at lower temperatures. Hot start PCR is a modified form of polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA by inactivating the DNA polymerase at lower temperatures. The polymerase chain reaction (PCR) is a method in molecular biology used to amplify a single copy or a few copies of specific pieces of DNA across several orders of magnitude, generating thousands to millions of copies of a target DNA sequence. In conventional PCR, the DNA polymerase is slightly active at room temperature, and to a lesser degree, even on ice. In some instances, when all the reaction components are put together, nonspecific primer annealing can occur at these low temperatures. This nonspecific annealed primer can then be extended by the DNA polymerase, generating nonspecific products and lowering product yields. The fundamental idea of hot start PCR is to reduce misprimed products during setup of the experiment. Early methods of hot start PCR involved excluding or limiting the concentration of one of the PCR reagents until the denaturation stage begins. This method later evolved into separating the reagents by covering one of the reagents in a wax which is solid at room temperature and dissolves during the initial stage in PCR. Hot start PCR significantly reduces nonspecific priming, the formation of primer dimers, and often, increases product yields. Classic methods, while effective, involve additional handling and increased risk of contamination. If the DNA polymerase is chemically modified, the re-activation time during the initial denaturation stage of the PCR cycle is increased because you need to wait for the enzyme to activate. This increased heating time could damage the DNA. Studies have also shown that some modifications by chemicals can cause issues for amplifying long strands of DNA (larger than 3kb). A disadvantage of using antibodies for hot start PCR is that you need a unique antibody per enzyme, which can be expensive if you are running a large number of PCRs. The antibodies are also derived from an animal cell, which could contaminate the results.