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CloningPCR Strategy

DNA Polymerase

With regard to PCR we only consider thermostable DNA Polymerases.

Taq DNA Polymerases

Taq DNA Polymerases is a thermostable enzyme from the thermophilic eubacterium Thermus aquaticus. It has a half-life of 40 min at 95°C. It is relatively cheap and is, therefore, the enzyme of choice for routine and control experiments. Taq DNA Polymerases produces PCR products with a one base 3'-overhang (most often being an A).

Taq DNA Polymerases is sold by many manufacturers and can also be obtained in the presence of a thermolabile inhibitor. During the initial denaturation step the inhibitor is denatured and active polymerase is released ("hot start"). This procedure improves the specificity of the PCR amplification by excluding the nonspecific reactions that occur at lower temperatures.

Proofreading DNA Polymerases

Proofreading or high fidelity DNA Polymerasess are enzymes with a very low rate of nucleotide misincorporation. The first discovered and best known enzyme is Pfu polymerase (from Pyrococcus furiosus). This enzyme incorporates approx. 767,000 nucleotides before making an error. In comparison Taq DNA Polymerases makes an error in approx. every 125,000 nucleotides. It is also one of the most thermostable DNA Polymerasess known. It can tolerate temperatures exceeding 95°C, enabling it to PCR amplify GC-rich targets. However, Pfu polymerase is more expensive and should, therefore, only be used when high accuracy is required. Pfu polymerase produces blunt-end PCR products.

Mixtures of polymerases

To amplify long DNA targets (5-35 kb) it is preferred to use a mixture of Taq and a proofreading DNA Polymerases. Optimised enzyme mixtures can be obtained from different manufacturers.