The discovery and development of high-fidelity polymerases has for many years been a key focus at New England Biolabs (NEB). Highfidelity amplification is essential for experiments whose outcome depends upon the correct DNA sequence (e.g., cloning, SNP analysis,
NGS applications). Whereas traditional fidelity assays are sufficient for Taq and other moderately faithful enzymes, Q5, an ultra highfidelity enzyme, pushes the limits of current methods used to assess this critical feature of DNA polymerases.
John A. Pezza, Ph.D., Rebecca Kucera, M.S.,
Luo Sun, Ph.D., New England Biolabs, Inc.
Introduction: What is fidelity?
The fidelity of a DNA polymerase is the result
of accurate replication of a desired template.
Specifically, this involves multiple steps, including
the ability to read a template strand, select the
appropriate nucleoside triphosphate and insert the
correct nucleotide at the 3´ primer terminus, such
that Watson-Crick base pairing is maintained. In
addition to effective discrimination of correct versus
incorrect nucleotide incorporation, some DNA
polymerases possess a 3´→5´ exonuclease activity.
This activity, known as “proofreading”, is used to
excise incorrectly incorporated mononucleotides
that are then replaced with the correct nucleotide.
High-fidelity PCR utilizes DNA polymerases that
couple low misincorporation rates with proofreading activity to give faithful replication of the
target DNA of interest.
When is fidelity important?
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.
How does a high-fidelity polymerase
ensure that the correct base is inserted?
High-fidelity DNA polymerases have several
safeguards to protect against both making and
propagating mistakes while copying DNA. Such
enzymes have a significant binding preference
for the correct versus the incorrect nucleoside
triphosphate during polymerization. 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 lag time increases the opportunity
for the incorrect nucleotide to dissociate before
polymerase progression, thereby allowing the
process to start again, with a correct nucleoside
triphosphate (1,2). If an incorrect nucleotide
is inserted, proofreading DNA polymerases
have an extra line of defense (Figure 1). The
perturbation caused by the mispaired bases is
detected, and the polymerase moves the 3´ end
of the growing DNA chain into a proofreading
3´→5´ exonuclease domain. There, the incorrect
nucleotide is removed by the 3´→5´ exonuclease
activity, whereupon the chain is moved back into
the polymerase domain, where polymerization
How is fidelity measured?
A variety of polymerase fidelity assays have been
described in the literature over the years, perhaps
the most famous being that of Thomas Kunkel (3).
The Kunkel method uses portions of the lacZαgene
in M13 bacteriophage to correlate host bacterial
colony color changes with errors in DNA synthesis.
Wayne Barnes built upon this assay and utilized
PCR to copy the entire lacZ gene and portions of
two drug resistance genes with subsequent ligation,
cloning, transformation and blue/white colony color
determination (4). In both assays, errors incorporated
in the lacZ gene cause a disruption in β-galactosidase
activity leading to a white colony phenotype. With
these lacZ-based experimental approaches, the
percentage of white colonies must be converted to
the number of errors per base incorporated. As a
more direct read-out of fidelity, Sanger sequencing of
individual cloned PCR products can also score DNA
polymerase fidelity and offers the advantage that all
mutations will be detected. Using this method, the
entire mutational spectrum of a polymerase can be
determined and there is no need to correct for nonphenotypic changes.
A modification of the lacZ Barnes assay is commonly
used at NEB for determination of DNA polymerase
fidelity, as the 1,000 amino acid open reading
frame affords a reasonable sequence window for
the scoring of DNA polymerase errors (Figure 2).
In this study, results from the lacZ assay were compared to Sanger sequencing to assess the fidelity of
Q5, a new NEB DNA polymerase.
In this study, Q5 was examined to determine its
fidelity compared to Taq DNA polymerase using
the two methods described below (Figure 2). A
3,874 bp target was PCR amplified with either
Taq (Thermopol Buffer), Q5 (Q5 Reaction Buffer
with or without GC enhancer) or Phusion®
(Phusion HF Buffer) DNA Polymerase. Observed mutation rates were determined using both the blue/
white selection method after 16 PCR cycles (4)
and by Sanger sequencing after 25 PCR cycles
(Table 1). The error rate per base incorporated
was determined after calculating the effective
number of amplification cycles for each experiment as described previously (4, 5). Comparing
the data sets from Taq indicates that the two
methods generate similar results with error rates
of ~1 in 3,500 bases. Q5, on the other hand,
yielded a significantly lower number of errors
than Taq in both assay systems, consistent with
an error rate of ~10-6. The side-by-side evaluation of Taq and Q5 using the blue/white method
suggests that Q5 is ~200X more faithful at replicating DNA than Taq. Similar results were observed for Q5 when the GC enhancer was added
to the reactions (data not shown). For Phusion,
the error rate was determined to be 80±39 times
better than Taq using the blue/white method and
84 times Taq using the sequencing method.