High-fidelity DNA Polymerases & When to use them

High-fidelity DNA Polymerases & When to use them

Taq polymerase is the tried and true standard when it comes to PCR, but that doesn’t mean it’s the most accurate polymerase for high-sensitivity applications. Instead, alternative enzymes, known as high-fidelity polymerases, offer higher accuracies in the amplicons resulting from PCR thanks to a combination of more stringent base reading and proofreading activities. While you don’t always need the improved accuracy that high-fidelity polymerases offer, having amplicons with perfect sequences can make a big difference when you plan to use your PCR products for downstream cloning or sequencing applications.

To help you decide if a high-fidelity polymerase is what your next experiment needs to succeed, let’s take a look at what exactly is meant by polymerase fidelity, how these enzymes work, and when high-fidelity polymerases offer advantages over Taq polymerase.

What is Polymerase Fidelity?

Polymerases aren’t perfect. As they’re speeding along a strand of DNA and adding in bases, they occasionally pair the wrong base with the template strand. While this mismatch might not be a big deal in a single replication reaction, in PCR the mistake can propagate through the amplification reactions until it is as or even more abundant than the correct version of the original sequence.

The error rate of a polymerase, which is the primary measure of fidelity, can be measured by a blue/white screening of lacZ clones or more directly by Sanger sequencing of individual cloned amplicons from a PCR reaction. Taq polymerase is particularly prone to making mismatch errors, with an error rate of over 200% when amplifying a 3-kB target sequence. That means that when amplifying a 3-kB sequence, every amplicon should contain at least two errors1 – and the mismatch rate only worsens for longer products.


What is a High-fidelity Polymerase?

The term high-fidelity describes any DNA polymerase that is less error-prone than Taq. While that might not seem impressive on its face – doing better than two mismatches per 3-kB product isn’t exactly a high bar – many of the high-fidelity polymerases currently on the market have error rates ranging from 10-50 times lower than Taq2. That means that for the same 3-kB target sequence, less than 5% of the amplicons will have any errors at all when using a high-fidelity polymerase.

The exact error rate of a high-fidelity polymerase mix depends on several factors. The standard high-fidelity polymerase is natural Pfu polymerase, purified from the thermophilic archaea Pyrococcus furiosus3. However, while highly accurate, Pfu polymerase is significantly slower at replication than Taq polymerase, so many high-fidelity PCR reagents include a mix of both Pfu and Taq polymerase to provide a balance of accuracy and speed. Engineered high-fidelity polymerases that modify the properties of Pfu polymerase to further improve its accuracy and increase its throughput are also available, but typically at a higher cost.

How do High-fidelity Polymerases work?

High-fidelity polymerases utilize two tricks that Taq polymerase lacks in order to reduce their mismatch rate during DNA replication.

First, the binding sites of these polymerases have a much higher affinity for the correct base than the binding site in Taq polymerase. This alone makes them more likely to insert the correct nucleotide to the end of the growing DNA strand. But even if the wrong nucleotide enters the polymerase initially, the sub-optimal architecture of the binding site for the mismatched nucleotide causes the extension reaction to proceed more slowly – thus allowing more time for the mistaken nucleotide to dissociate from the polymerase and the extension process to begin again with the correct nucleotide3,4.

Second, high-fidelity polymerases are also capable of proofreading as they move along the DNA strand. A second domain of the enzyme serves as a 3’à5’ exonuclease that is activated when the structural perturbation caused by a mismatch is detected. This exonuclease domain allows the polymerase to move backwards along the replicating strand, remove the mismatched base, and restart the extension reaction with the correct base5.

When do you need a High-fidelity Polymerase?

The increased accuracy of high-fidelity polymerases makes them much more suitable for sensitive downstream applications than standard Taq polymerase. Using PCR to generate enough DNA mass for sequencing is a common application where high-fidelity polymerases are preferred, since the presence of mismatches in amplicons from Taq PCR can influence the analysis of your sequencing data. The importance of using high-fidelity polymerases scales according to the length of your amplicon since Taq polymerase will produce correspondingly more errors in a longer target sequence. Generating amplicons for cloning protein-coding sequences is also a good time to consider high-fidelity polymerases since substitution mutations can inactivate or alter the expressed protein.

More broadly, high-fidelity polymerases can be used for any application for which you would ordinarily use Taq polymerase. However, in applications where the presence of DNA mismatches is not significant – for example, checking for the presence or absence of an insert in a clone – it is worth noting that high-fidelity polymerase is typically more expensive than Taq polymerase and requires a longer total PCR cycle time due to the slower extension rates of proofreading polymerases.


High-fidelity DNA polymerases offer a highly accurate alternative to standard Taq polymerase when using PCR to produce amplicons for sensitive downstream applications such as cloning and sequencing. When choosing a high-fidelity polymerase, be sure to compare error rates and throughput rates to find the balance of accuracy, speed, and cost that is right for your experiments.


1PCR Fidelity Calculator. Thermo Fisher. www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/pcr-fidelity-calculator.html

2Cline J, Braman JC, Hogrefe HH. 1996. Nucleic Acids Research 24(18):3546-3551.

3McInerney P, Adams P, Hazi MZ. 2014. Molecular Biology International 2014:287430.

4Joyce CM, Benkovic SJ. 2004. Biochemistry 43(45):14317-14324.

5Reha-Krantz LJ. 2010. Biochimica et Biophysica Acta – Proteins and Proteomics 1804(5):1049-1063.


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