How is Fidelity Measured?

Return to High-Fidelity PCR

Fidelity comparisons between polymerases can be expressed in absolute terms, often by the number of errors per 1,000 or 10,000 nucleotides, or expressed as the number of theoretical errors per base. The level of fidelity can also be expressed in relative terms, often using Taq DNA polymerase as the relative standard (Table 1). Note that while single base substitution errors are the easiest to assess, polymerases are also capable of insertion/ deletion errors that can lead to frameshift mutations, a highly deleterious type of error.

Comparison of High-Fidelity Polymerases

(Reported by supplier)
(For simple templates4)
(For complex templates4)
Q5 High-Fidelity
DNA Polymerase (NEB)
~280X Taq1 20 kb simple;
10 kb complex
10 s/kb 10 s/kb (<1 kb)
20–30 s/kb (>1 kb)
Phusion High-Fidelity
DNA Polymerase* (NEB)
39X Taq1 20 kb simple;
10 kb complex
15 s/kb 30 s/kb
AccuPrime™ Pfx (Life) 26X Taq2 12 kb3 60 s/kb3
PfuUltra™ II Fusion HS
20X Taq2 19 kb3
15 s/kb (<10 kb3)
30 s/kb (>10 kb3)
PfuUltra High-Fidelity
DNA Polymerase (Agilent)
19X Taq2 17 kb simple;
6 kb complex
60 s/kb (<10 kb)
120 s/kb (>10 kb)
60 s/kb (<6 kb)
120 s/kb (>6 kb)
KOD DNA Polymerase
12X Taq1 6 kb simple;
2 kb complex
10–20 s/kb 30–60 s/kb
Platinum® Taq HiFi (Life) 6X Taq2 20 kb3 60 s/kb3

We continue to investigate assays to characterize Q5's very low error rate to ensure that we present the most accurate fidelity data possible (Potapov, V, and Ong, J.L. (2017) PloS ONE, 12(1): e0169774.).

2 PCR-based mutation screening in lacZ (NEB), lacI (Agilent) or rpsL (Life).

* Phusion DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific.

Template not specified.

Simple templates include plasmid, viral and E. coli genomic DNA. Complex templates include plant, human and other mammalian genomic DNA.

Values provided by individual manufacturers.

Polymerase fidelity assays take many forms and have been used extensively for comparing high-fidelity polymerases. The pioneering work of Thomas Kunkel (1) utilized portions of the lacZα gene in M13 bacteriophage to correlate host bacterial colony color changes with errors in DNA synthesis, using a variety of prepared M13 substrates for assessing single base nucleotide substitution errors or frameshift mutations. William Thilly (2) championed denaturing gradient gel electrophoresis to distinguish between heteroduplexes formed when error-containing PCR amplicon strands reannealed in the presence of an excess of wild type (WT) complementary strands (lower melting temperature), and the corresponding WT homoduplex form (higher melting temperature).

Another approach, championed by Wayne Barnes (3), utilizes 16 cycles of 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. Similar to the Kunkel assays, most errors in the lacZ-encoding β-galactosidase gene cause a loss-of-function of the ability to utilize the Xgal substrate on agar plates that would normally result in a blue colony. This assay is presently used at New England Biolabs for large scale, relatively quick determination of DNA fidelity, as the 3,874 bp amplicon affords a reasonable sequence space for the scoring of DNA polymerase errors. Sanger sequencing of individual cloned PCR products can also score fidelity of the thermophilic DNA polymerase employed in the PCR as long as the error rate of the polymerase used for sequencing is lower than that of the PCR polymerase.


(1) Kunkel, T.A. and Tindall, K.R. (1987) Biochemistry, 27, 6088–6013.
(2) Ling, L.L. et al. (1991) Genome Research, 1, 63–69.
(3) Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147.