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 technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In hot start PCR specific antibodies or reversible chemical modifications of the protein (usually modifications of the lysine with organic acid anhydride) are using are used to block the activity of the DNA polymerase at lower temperature. An initial activation step at 95? is required for activation of the protein. This step will both denature antibodies linked to the active center of the enzyme, and also remove any lysine modifications made with acid anhydride. The anti-Taq antibodies reduce the Taq polymerase activity below 72?, the optimal temperature at which the enzyme extends the primers. When the specific antibodies detach from Taq-polymerase, the amplification proceeds with greater specificity.
In conventional PCR, the DNA polymerase is modestly 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 due to these low temperatures. This nonspecific annealed primer can then be extended by the DNA polymerase, generating nonspecific products and lowering product yields.
Hot start PCR significantly reduces nonspecific priming, the formation of primer dimers, and often, increases product yields. In Hot start long and accurate PCR, the impact on yield can be dramatic. Classic methods, while effective, involve additional handling and increased risk of contamination.
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