The finest PCR fidelity in over 10 years
In this blog post we look at Q5® High-Fidelity DNA Polymerase by describing:
- What is fidelity or “Proofreading activity” of a Polymerase?
- When is fidelity important? Eller In which applications are fidelity important?
- Why is Q5 so proofreading and robust?
➡️ Right now, you save 20% on Q5 Products from NEB
What is fidelity or “Proofreading activity” of a Polymerase?
DNA polymerase fidelity describes the replication accuracy of a desired template during DNA amplification, also named “Proofreading” activity. High-fidelity DNA polymerases have several checkpoints to protect against making and propagating mistakes while copying DNA:
1. Precision of template strand reading
2. Correctness of nucleoside triphosphates inserted
3. Replacement of incorrect nucleotides incorporated
The precision of template strand reading
Precision reading of the template strand means that the polymerase enzyme interprets the exact nucleoside triphosphates order exposed on the DNA strand to be replicated. This ability of the fidelity polymerases is obtained by a significant binding preference for the correct versus the incorrect nucleotide triphosphate during the DNA polymerization.
Correctness of nucleoside triphosphates inserted
Correctness of nucleoside triphosphates inserted is ensured by selecting and inserting the correct nucleoside triphosphates at the 3´-primer terminus in accordance with Watson-Crick base pairing. 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 time increases the opportunity for the incorrect nucleotide to dissociate before incorporation, thereby allowing the process to start again (and for a correct nucleotide triphosphate to bind) (Ref. 1,2).
Replacement of incorrect nucleotides incorporated
Finally, the replacement of incorrect nucleotides incorporated is checked by effective discrimination of correct versus incorrect nucleotide incorporation. If an incorrect nucleotide is inserted, proofreading DNA polymerases have an extra line of defense. They can "sense" the perturbation caused by the mispaired bases and move the 3' end of the growing DNA chain into a proofreading 3'→5' exonuclease domain. The incorrect nucleotide is removed by the 3'→5' exonuclease activity before the chain is moved back into the polymerase domain, where polymerization can continue with the correct nucleotide. (See Figure 1.)
Figure 1. DNA Replication with a Proofreading Polymerase
Extension proceeds along the template strand at the 3' end of the newly synthesized strand. When the polymerase recognizes an error, the mismatched base is transferred to the exonuclease active site, and the base is excised. The extended strand returns to the polymerase domain, re-anneals to the template strand, and replication continues.
When is fidelity important?
Fidelity is important for applications in which the DNA sequence must be correct after the amplification, including:
- Cloning/subcloning protein expression
- SNP (Single Nucleotide Polymorphism) analysis
- RNA analysis by RT-PCR
- Next generation sequencing applications
On the other hand, fidelity is less important for many diagnostic applications where the read-out is simply the presence or absence of a product.
Why is Q5 so proofreading and robust?
New England Biolabs (NEB) has been involved in the discovery and development of high-fidelity polymerases for many years. Beginning with the discovery of Vent® DNA Polymerase from Thermococcus litoralis in 1990. Since then, NEB researchers have identified and engineered several high-fidelity polymerases.
In 2012, the Q5 DNA polymerase was released by NEB as a new standard for both fidelity and robust performance. The fidelity of Q5, measured as >280 times* higher than Taq polymerase, ensures ultra-low error rates. In addition, Q5 demonstrates:
Robust amplification with minimal optimization
Superior coverage for a broad range of amplicons, regardless of GC content
Shorter PCR protocols
Amplify templates up to 20 kb
* 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.
The superior proofreading activity of Q5 DNA polymerase is caused by a fusion of to the processivity-enhancing Sso7d DNA binding domain polymerase from Sulfolobus solfataricus; a species of thermophilic archaeon isolated and discovered in the Solfatara volcano. This fusion improves fidelity (more than 50-fold higher than Taq), but also speed and performance reliability (amplifying targets up to 20 kb long with extension times as low as 10 seconds per kb). In addition, the Q5 buffer system has been extensively optimized to provide robust performance across both high-GC and high-AT regions, as well as other challenging amplicons.
1 PCR-based mutation screening in lacZ (NEB), lacI (Agilent) or rpsL (Life)
2 Due to the very low frequency of misincorporation events being measured, the error rate of high-fidelity enzymes like Q5 is difficult to measure in a statistically significant manner. Although measurements from assays done side-by-side with Taq yield a Q5 fidelity values of approximately 200X Taq, we report “>100X Taq” as a conservative value.
3 Takagi et al (1997) Appl. Env. Microbiol. 63, 4504-4510.
Today NEB also provides:
Q5U® Hot Start High-Fidelity DNA Polymerase (NEB #M0515). Q5U is a modified version of Q5 High-Fidelity DNA Polymerase containing a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases. This is useful for amplifying bisulfite-converted, enzymatically deaminated, or damaged DNA, preventing carryover contamination in PCR (when used with dUTP and UDG), and in USER cloning methods. Learn more Q5U® Hot Start High-Fidelity DNA Polymerase here.
Q5 Blood Direct 2X Master Mix (NEB #M0500) can amplify a wide variety of targets direct from dried blood spots or up to 30% whole human blood, skipping DNA purification.
Explore Q5® yourself!
Explore or challenge the outstanding Q5 fidelity features of the Q5 products, by requesting a Free-of-Charge sample or buy now and save 20% on all Q5 products from NEB.
Save 20% on Q5 products from NEB
1) Johnson, K. A. (2010) Biochimica et Biophysica Acta, 1804, 1041–1048. PMID: 20079883
2) Joyce, C. M., & Benkovic, S. J. (2004) Biochemistry, 43, 14317–14324. PMID: 15533035