Q5® High-Fidelity DNA Polymerases

Fidelity at its Finest.

Q5® High-Fidelity DNA Polymerase (NEB #M0491) sets a new standard for both fidelity and robust performance. With the highest fidelity amplification available (>280 times higher than Taq), 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. Q5 master mixes contain dNTPs, Mg++ and a proprietary broad-use buffer requiring only the addition of primers and DNA template for robust amplification, regardless of GC content.

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 about this product.

NEW: 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. Learn more about this product.

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Comparison of High-Fidelity Polymerases

1 We continue to investigate improved 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 lacI (Agilent) or rpsL (Life).


  • Highest fidelity amplification (>280X higher than Taq)
  • Ultra-low error rates
  • Superior performance for a broad range of amplicons (from high AT to high GC)
  • Hot start and master mix formats available

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 (NEB #B9027) 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.

Robust Amplification with Q5 (A) and Q5 Hot Start (B) High-Fidelity DNA Polymerases

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.

In contrast to chemically modified or antibody-based hot start polymerases, NEB's Q5 Hot Start (NEB #M0493) 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.

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.

Master Mix and Stand-Alone Formats Provide Convenience and Flexibility

Q5® is a registered trademark of New England Biolabs, Inc.
LabChip® is a registered trademark of Caliper Life Sciences, part of Perkin Elmer, Inc.

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FAQs for Q5® High-Fidelity DNA Polymerases
Protocols for Q5® High-Fidelity DNA Polymerases
Application Notes for Q5® High-Fidelity DNA Polymerases
    Publications related to Q5® High-Fidelity DNA Polymerases
    • Vladimir Potapov, Jennifer L. Ong. (2017) Examining Sources of Error in PCR by Single-Molecule Sequencing. PLOS One; PubMedID: 28683110
    • Amin Zargar, David N Quan, Milad Emamian, Chen Yu Tsao, Hsuan-Chen Wu, Chelsea R Virgile, William E Bentley (2015) Rational design of 'controller cells' to manipulate protein and phenotype expression. Metab Eng; PubMedID: 25908186, DOI: 10.1016/j.ymben.2015.04.001
    • Yuan Xue, Jossef Osborn, Anand Panchal, Jay L Mellies (2015) The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc. Appl Environ Microbiol; 81, 3766-74. PubMedID: 25819956, DOI: 10.1128/AEM.00507-15
    • Longhai Dai, Can Liu, Yueming Zhu, Jiangsheng Zhang, Yan Men, Zeng Yan, Yuanxia Sun (2015) Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii. Plant Cell Physiol; PubMedID: 25759326, DOI: 10.1093/pcp/pcv043
    • Jun Wu, Daiji Okamura, Mo Li, Keiichiro Suzuki, Chongyuan Luo, Li Ma, Yupeng He, Zhongwei Li, Chris Benner, Isao Tamura, Marie N Krause, Joseph R Nery, Tingting Du, Zhuzhu Zhang, Tomoaki Hishida, Yuta Takahashi, Emi Aizawa, Na Young Kim, Jeronimo Lajara, Pedro Guillen, Josep M Campistol, Concepcion Rodriguez Esteban, Pablo J Ross, Alan Saghatelian, Bing Ren, Joseph R Ecker, Juan Carlos Izpisua Belmonte (2015) An alternative pluripotent state confers interspecies chimaeric competency. Nature; PubMedID: 25945737, DOI: 10.1038/nature14413
    • Harish Nag Kankipati, Marta Rubio-Texeira, Dries Castermans, George Diallinas, Johan M Thevelein (2015) Sul1 and Sul2 Sulfate Transceptors Signal to Protein Kinase A upon Exit of Sulfur Starvation. J Biol Chem; 290, 10430-46. PubMedID: 25724649, DOI: 10.1074/jbc.M114.629022
    • Yonghe Zhang, Huiming Huang, Shanshan Xu, Bo Wang, Jianhua Ju, Huarong Tan, Wenli Li (2015) Activation and enhancement of Fredericamycin A production in deepsea-derived Streptomyces somaliensis SCSIO ZH66 by using ribosome engineering and response surface methodology. Microb Cell Fact; 14, 64. PubMedID: 25927229, DOI: 10.1186/s12934-015-0244-2
    • Yafeng Li, Delu Song, Ying Song, Liangliang Zhao, Natalie Wolkow, John W Tobias, Wenchao Song, Joshua L Dunaief (2015) Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium. J Biol Chem; 290, 11918-34. PubMedID: 25802332, DOI: 10.1074/jbc.M115.645903
    • Christine Henke, Pamela L Strissel, Maria-Theresa Schubert, Megan Mitchell, Claus C Stolt, Florian Faschingbauer, Matthias W Beckmann, Reiner Strick (2015) Selective expression of sense and antisense transcripts of the sushi-ichi-related retrotransposon - derived family during mouse placentogenesis. Retrovirology; 12, 9. PubMedID: 25888968, DOI: 10.1186/s12977-015-0138-8
    • Binyamin D Berkovits, Christine Mayr (2015) Alternative 3' UTRs act as scaffolds to regulate membrane protein localization. Nature; PubMedID: 25896326, DOI: 10.1038/nature14321
    • Bert De Rybel, Milad Adibi, Alice S. Breda, Jos R. Wendrich, Margot E. Smit, Ondej Novk, Nobutoshi Yamaguchi, Saiko Yoshida, Gert Van Isterdael, Joakim Palovaara, Bart Nijsse, Mark V. Boekschoten, Guido Hooiveld, Tom Beeckman, Doris Wagner, Karin Ljung, Christian Fleck, Dolf Weijers (2014) Integration of growth and patterning during vascular tissue formation in Arabidopsis Science; 345, 1255215. PubMedID: 25104393, DOI: 10.1126/science.1255215
    • Xin Duan, Arjun Krishnaswamy, Irina De la Huerta, Joshua R Sanes (2014) Type II Cadherins Guide Assembly of a Direction-Selective Retinal Circuit. Cell; 158, 793-807. PubMedID: 25126785, DOI: 10.1016/j.cell.2014.06.047
    • Martin Kostovcik, Craig C Bateman, Miroslav Kolarik, Lukasz L Stelinski, Bjarte H Jordal, Jiri Hulcr (2014) The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing. ISME J; PubMedID: 25083930, DOI: 10.1038/ismej.2014.115
    • Connelly CM1, Porter LR2, TerMaat JR (2014) PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target BMC Med Genet; 15, 130. PubMedID: 25495904, DOI: 10.1186/s12881-014-0130-5
    • Vidhyadhar Nandana, Sushant Singh, Abhay Narayan Singh, Vikash Kumar Dubey (2014) Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence. Protein Expr Purif; 103C, 16-22. PubMedID: 25173974, DOI: 10.1016/j.pep.2014.08.003
PCR Selection Tool
Choose from one of the largest selections of polymerases for PCR applications from the leader in enzyme technology and bring unparalleled confidence to your experiments.
Comparison of High-Fidelity Polymerases
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.
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
DNA Polymerase Selection Chart
NEB offers a guidelines for choosing the correct DNA polymerase for your application by providing a list of specific properites.
Several factors govern which polymerase should be used in a given application, including: 

Template/product specificity: Is RNA or DNA involved? Is the 3´ terminus at a gap, nick or at the end of the template? 

Removal of existing nucleotides: Will the nucleotide(s) be removed from the existing polynucleotide chain as part of the protocol? If so, will they be removed from the 5´ or the 3´ end? 

Thermal stability: Does the polymerase need to survive incubation at high temperature or is heat inactivation desirable? 

Fidelity: Will subsequent sequence analysis or expression depend on the fidelity of the synthesized products?

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Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.