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Home > Product Overview > Q5® High-Fidelity DNA Polymerase
 
Q5® High-Fidelity DNA Polymerase
Fidelity at its finest: > 100X higher fidelity than Taq
 
Q5 Fidelity Graph
Comparison of high fidelity polymerases
Q5 High-Fidelity DNA Polymerase sets a new standard for both fidelity and performance. With the highest fidelity amplification available (>100X higher than Taq), Q5 DNA Polymerase results in ultra-low error rates.
 
Figure 1: Comparison of high fidelity polymerases
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.


Q5 High-Fidelity DNA Polymerase

Q5 High-Fidelity DNA Polymerase sets a new standard for both fidelity and robust performance. With the highest fidelity amplification available (>100 times higher than Taq) (Table 1), Q5 DNA Polymerase results in ultra-low error rates. Q5 DNA Polymerase is composed of a novel polymerase that is fused to the processivity-enhancing Sso7d DNA binding domain, improving speed, fidelity and reliability of performance.

The Q5 buffer system is designed to provide superior performance with minimal optimization across a broad range of amplicons, regardless of GC content. For routine or complex amplicons up to ~65% GC content, Q5 Reaction Buffer provides reliable and robust amplification. For amplicons with high GC content (>65% GC), addition of the Q5 High GC Enhancer ensures continued maximum performance. Q5 and Q5 Hot Start DNA Polymerases are available as standalone enzymes, or in a master mix format for added convenience. Master mix formulations include dNTPs, Mg++ and all necessary buffer components.


Q5 Hot Start High-Fidelity DNA Polymerase


In contrast to chemically modified or antibody-based hot start polymerases, NEB's Q5 Hot Start utilizes a unique synthetic aptamer. This molecule binds to the polymerase through non-covalent interactions, blocking activity during the reaction setup. The polymerase is activated during normal cycling conditions, allowing reactions to be set up at room temperature. Q5 Hot Start does not require a separate high temperature activation step, shortening reaction times and increasing ease-of-use. Q5 Hot Start Polymerase is an ideal choice for high specificity amplification and provides robust amplification of a wide variety of amplicons, regardless of GC content.
 


Five quality features of Q5

1 Fidelity – the highest fidelity amplification available (>100X higher than Taq)
2 Robustness – high specificity and yield with minimal optimization
3 Coverage – superior performance for a broad range of amplicons (from high AT to high GC)
4 Speed – short extension times
5 Amplicon length – robust amplifications up to 20 kb for simple templates, and 10 kb for complex


Q5 DNA Polymerases set the new standard in both fidelity and performance

Figure 2: Robust amplification with Q5 (A) and Q5 Hot Start (B) High-Fidelity DNA Polymerases, regardless of GC content

Amplification of a variety of human genomic amplicons from low to high GC content using either Q5 or Q5 Hot Start
High-Fidelity DNA Polymerase. Reactions using Q5 Hot Start were set up at room temperature. All reactions were
conducted using 30 cycles of amplification and visualized by microfluidic LabChip® analysis.



Q5 DNA Polymerases outperform other commercially available high fidelity polymerases

Table 1: Comparison of high fidelity polymerases
PRODUCT NAME
(Supplier)
POLYMERASE FIDELITY
(Reported by supplier)
MAXIMUM AMPLICON
LENGTH6
EXTENSION TIME6
(For simple templates5)
EXTENSION TIME6
(For complex templates5)
Q5 High-Fidelity
DNA Polymerase (NEB)
>100X Taq1,2 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)
>50X Taq1,2 20 kb simple;
10 kb complex
15 s/kb 30 s/kb
AccuPrime™ Pfx (Life) 26X Taq1 12 kb4 60 s/kb4
PfuUltra™ II Fusion HS
(Agilent)
20X Taq1 19 kb4 15 s/kb (<10 kb4)
30 s/kb (>10 kb4)
PfuUltra High-Fidelity
DNA Polymerase (Agilent)
19X Taq1 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)
Platinum® Taq HiFi (Life) 6X Taq1 20 kb4 60 s/kb4
KOD DNA Polymerase
(EMD)
4X Taq3 6 kb simple;
2 kb complex
10–20 s/kb 30–60 s/kb


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 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.
4 Template not specified.
5 Simple templates include plasmid, viral and E. coli genomic DNA. Complex templates include plant, human and other mammalian
genomic DNA.
6 Values provided by individual manufacturers.


Figure 3: Q5 DNA Polymerase offers superior amplification for a wide range of templates, regardless of GC content

Amplification of a variety of human genomic amplicons from low to high GC content demonstrates the broad performance of Q5 High-
Fidelity DNA Polymerase. All reactions were conducted using 20 ng of input template and included 30 cycles of amplification. Results
were visualized by microfluidic LabChip® analysis. Competitor polymerases were cycled according to manufacturer's recommendations.
For the final three amplicons, GC Buffers or enhancers were used when supplied with the polymerase.



Figure 4: Q5 Hot Start DNA Polymerase offers superior amplification for a wide range of templates, regardless of GC content

Amplification of a variety of human genomic amplicons from low to high GC content demonstrates the broad performance of Q5 Hot Start High-Fidelity
DNA Polymerase. All reactions were set up at room temperature using 20 ng of input template and included 30 amplification cycles. Results were visualized
by microfluidic LabChip® analysis. Competitor polymerases were cycled according to manufacturer's recommendations. For the final three amplicons, GC
Buffers or enhancers were used when provided with the polymerase.



Figure 5: Amplification Performance Across a Wide Range of Genomic Targets

PCR was performed with a variety of amplicons, with GC content ranging from high AT to high GC, with Q5 and several other commercially available polymerases. All polymerases were cycled according to manufacturer's recommendations, including use of GC Buffers and enhancers when recommended. Yield and purity of reaction products were quantitated and represented, as shown in the figure key, by dot color and size. A large dark green dot represents the most successful performance. Q5 provides superior performance across the range of GC content.


Q5 DNA Polymerases are available in master mix and hot start formulations

 
Products
  


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. Examples of relative error rates are shown in Table 3. 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.

Polymerase fidelity assays take many forms and have been used extensively for comparing high-fidelity polymerases. The pioneering work of Thomas Kunkel (5) utilized portions of the lacZa 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 (6) 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 (7), 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 ß-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.

5. Kunkel, T.A. and Tindall, K.R. (1987) Biochemistry, 27, 6088–6013.
6. Ling, L.L. et al. (1991) Genome Research, 1, 63–69.
7. Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147.
Technical Resource Quick Links
 
IPAD® and MACBOOK AIR® are registered trademarks of Apple Computer, Inc.
PHUSION® is a registered trademark and property of Thermo Fisher Scientific.
Phusion® DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific.
ACCUPRIME™ is a trademark of Life Technologies, Inc.
PLATINUM® is a registered trademark of Life Technologies, Inc.
PFUULTRA™ is a trademark of Agilent Technologies, Inc.
Q5® and VENT® are registered trademarks of New England Biolabs, Inc.
DEEP VENT™ and 9°N™ are trademarks of New England Biolabs, Inc.
LABCHIP® is a registered trademark of Caliper Life Sciences, part of Perkin Elmer, Inc.





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