DNA polymerases for High-Fidelity PCR
High fidelity PCR is frequently used for cloning and mutagenesis. However standard Taq polymerase is not sutable for this reaction, since it simply makes to many mistakes during amplification.
High-Fidelity DNA Polymerases are important for applications in which the DNA sequence needs to be correct after amplification. Fidelity is important for applications in which the DNA sequence must be correct after amplification. Common examples include cloning/subcloning DNA for protein expression, SNP analysis and next generation sequencing applications. Fidelity is less important for many diagnostic applications where the read-out is simply the presence or absence of a product.
The fidelity of a polymerase refers to its ability to insert the correct base during PCR. Conversely, the rate of misincorporation is known as a polymerase's error rate. High-fidelity PCR, utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest.
Different types of high-fidelity DNA polymerases are distinguished, including Pfu polymerase, a naturally faithful DNA polymerase due to its 3'-5 'exonuclease activity as well as modified DNA polymerases to obtain higher fidelity capacities.