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 taq 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 are used to block the Taq-polymerase at lower temperature. An initial step at 95℃ is required for denaturing the antibodies linked to the active center of the enzyme. 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 detaches from Taq-polymerase the amplification proceeds with greater specificity.
In conventional PCR, the Taq DNA polymerase is 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 Taq 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. [1]
References
- ↑ Primrose, S. B.; Richard M. Twyman; R. W. Old (2001). Principles of gene manipulation. Wiley-Blackwell. p. 23. ISBN 978-0-632-05954-6.