Hot Start PCR - HotStart Polymerase Chain Reaction
Hot Start PCR : An Introduction
Updated April 2011 - Hot Start PCR allows the inhibition of polymerase activity during PCR reaction preparation. By limiting polymerase activity prior to PCR cycling, Hot Start PCR reduces non-specific amplification and increases PCR product target yield.
Hot Start PCR is commonly performed by using included chemical modifications, wax-barrier methods, and inhibition by a taq-directed antibody.
PCR or polymerase chain reaction uses DNA polymerases which were isolated from thermostable organisms such as the Taq enzyme, a eubacterial type I DNA polymerase, and Pfu, an archaeal type B DNA polymerase.
PCR amplifies DNA by multiple cycles of melting DNA at high temperature, annealing primers at a lower temperature, and then allowing polymerase extension at an intermediate temperature. Unfortunately, these thermophilic DNA polymerases show a very small but measurable polymerase activity at room temperature during assembly of the experiment.
This in-efficient DNA polymerase activity will catalyze the extension of any annealed 3' ends. After PCR amplification, the resulting product contains a mixture of specific and non-specific bands. Moreover, the 5'-3' exonuclease activity of the DNA polymerasel degrades any free 5' ends of partially annealed nucleic acids, destroying the primer and template substrates of the polymerase reaction. This results in a lower yield of the desired pcr product.
Hot Start PCR : Publications
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