Q5^® high-fidelity DNA polymerase

What is fidelity or “Proofreading activity” of a Polymerase? 

DNA polymerase fidelity describes the replication accuracy of a desired template during DNA amplification, also named “proofreading” activity. High-fidelity DNA polymerases have several checkpoints to protect against making and propagating mistakes while copying DNA: 

1. Precision of template strand reading  
2. Correctness of nucleoside triphosphates inserted  
3. Replacement of incorrect nucleotides incorporated  

1. The precision of template strand reading 
Precision reading of the template strand means that the polymerase enzyme interprets the exact nucleoside triphosphates order exposed on the DNA strand to be replicated. This ability of the fidelity polymerases is obtained by a significant binding preference for the correct versus the incorrect nucleotide triphosphate during the DNA polymerization.

2. Correctness of nucleoside triphosphates inserted
Correctness of nucleoside triphosphates inserted is ensured by selecting and inserting the correct nucleoside triphosphates at the 3'-primer terminus in accordance with Watson-Crick base pairing. If an incorrect nucleotide does bind in the polymerase active site, incorporation is slowed due to the sub-optimal architecture of the active site complex. This time increases the opportunity for the incorrect nucleotide to dissociate before incorporation, thereby allowing the process to start again (and for a correct nucleotide triphosphate to bind) (ref. 1 and 2).

3. Replacement of incorrect nucleotides incorporated
Finally, the replacement of incorrect nucleotides incorporated is checked by effective discrimination of correct versus incorrect nucleotide incorporation. If an incorrect nucleotide is inserted, proofreading DNA polymerases have an extra line of defense.

They can "sense" the perturbation caused by the mispaired bases and move the 3' end of the growing DNA chain into a proofreading 3'→5' exonuclease domain. The incorrect nucleotide is removed by the 3'→5' exonuclease activity before the chain is moved back into the polymerase domain, where polymerization can continue with the correct nucleotide (see Figure 1).

Figure 1. DNA replication with a proofreading polymerase 
Extension proceeds along the template strand at the 3' end of the newly synthesized strand. When the polymerase recognizes an error, the mismatched base is transferred to the exonuclease active site, and the base is excised. The extended strand returns to the polymerase domain, re-anneals to the template strand, and replication continues.