Alkami Quick Guide™ for PCR

PCR Polymerases

Each thermophilic polymerase has unique characteristics, such as pH and salt optima, that affect the efficacy of your PCR protocol. As applications of PCR become increasingly sophisticated and specific, the special needs of your experiments can utilize these distinctive properties.

A high fidelity polymerase typically demonstrates 3' to 5' exonuclease activity, that is, a "proofreading" ability where the polymerase can correct misincorporated nucleotides in the strand of DNA being synthesized. Several optimized polymerase mixtures have been developed which combine a standard polymerase, such as Taq, with a small amount of a high fidelity polymerases, such as Pfu, Vent, and Deep Vent.

A significant problem with PCR is the lack of fidelity which occurs under various conditions with different polymerases. Polymerase errors can take place during five distinct activities of the extension phase: the binding of the dNTP by the polymerase, the rate of phosphodiester bond formation; the rate of pyrophosphate release; the continuation of extension after a misincorporation; and the degree of 3’-5’ exonuclease proofreading activity.

There are several reasons for the variation of error rates in addition to those involved in the laxity of the polymerase. One potential cause could be physical damage to the DNA resulting in misincorporated bases, gaps and/or crossover products. The condition of the template can also affect fidelity. This is something that is not generally considered when error rates are determined and compared.

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The error rates for the various polymerases are defined by errors per nucleotide. Several studies have determined that polymerase error rates can range between 2.1 x 10-4 and 1.6 x 10-6 errors per nucleotide per extension.

Additionally, the types of errors that occur during the PCR are dependent on the particular DNA polymerase. For example, among high-fidelity (proofreading) polymerases, there is a tendency to degrade single-stranded DNA primer one base at a time from the 3' end during the course of an experiment. As a result, some of the positions on the primer could be degraded past the positions containing the desired base changes before the primer anneals to the template. Note that longer primers have a higher initial degradation rate than shorter primers.

The following DNA polymerase characteristics were collected from a variety of sources and thus under different conditions. They are meant only as guidelines for tuning your PCR protocols.