DNA Replication with a Proofreading Polymerase

Learn how proofreading polymerases recognize and correct mismatched bases.


Proofreading polymerases have several checkpoints to prevent incorrect nucleotide incorporation during the DNA extension process. 

The first checkpoint is a result of the enzyme significant binding preference for the correct versus incorrect nucleoside triphosphate during polymerization. If an incorrect base does bind to the active site, incorporation is slowed, increasing the opportunity for the incorrect nucleotide to dissociate and a correct nucleotide to bind.

Alternatively, if an incorrect nucleotide is incorporated, the proofreading polymerase detects the perturbation caused by the mispaired bases and shifts the three prime end of the growing strand to the polymerase's proofreading exonuclease active site domain, where the three prime to five prime exonuclease activity removes the mispaired base. With the mispaired base removed, the polymerase shifts the strand back into the polymerase domain and continues adding bases and extending the DNA.

Fidelity is critical for many applications, including cloning and next generation sequencing, and NEB has developed a broad portfolio of high fidelity polymerases for use in PCR.

Visit ConfidentPCR.com for help in selecting the right DNA polymerase for your experiment.

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