The fidelity of a polymerase refers to its ability to insert the correct base during PCR. Conversely, the rate of misincorporation is known as a polymerase’s error rate. High-fidelity PCR, utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest. NEB scientists were the first to identify and commercialize a high-fidelity DNA polymerase suitable for PCR, namely Vent® DNA Polymerase. Since its discovery, several other high fidelity polymerases have been discovered and engineered – all with differing processivity, speed and levels of accuracy.
FAQs for High-Fidelity PCR
Protocols for High-Fidelity PCR
- Guidelines for PCR Optimization for Deep Vent® DNA Polymerase
- PCR Optimization (E0555)
- PCR Optimization of the Control Template using Phusion® High-Fidelity PCR Kit
- PCR Optimization with Phusion® High-Fidelity PCR Kit
- PCR Protocol for Crimson LongAmp™ Taq DNA Polymerase (M0326)
- PCR Protocol for Phusion® Hot Start Flex DNA Polymerase (M0535)
- PCR Protocol Phusion® DNA Polymerase
- PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (M0541)
- PCR Using Q5® High-Fidelity DNA Polymerase (M0491)
- PCR Using Q5® Hot Start High-Fidelity DNA Polymerase (M0493)
- Protocol for a Routine Deep Vent® (exo-) PCR
- Protocol for a Routine Deep Vent® PCR
- Protocol for a Routine PCR (E0555)
- Protocol for a Routine PCR with Phusion® High-Fidelity PCR Kit
- Protocol for Phusion® High-Fidelity PCR Master Mix with GC Buffer
- Protocol for Phusion® Hot Start Flex 2X Master Mix
- Protocol for Q5® High-Fidelity 2X Master Mix
- Protocol for Q5® Hot Start High-Fidelity 2X Master Mix
- Protocol Phusion® High-Fidelity PCR Master Mix with HF Buffer
Anatomy of a Polymerase - How Structure Effects Function
Polymerase Fidelity: What is it, and what does it mean for your PCR?
Understanding Variability in DNA Amplification Reactions
- PCR Selector
- DNA Polymerase Selection Chart
- PCR Troubleshooting Guide
- Guidelines for PCR Optimization with Thermophilic DNA Polymerases
Other Tools & Resources
- 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, PubMedID: 25888968, DOI: 10.1186/s12977-015-0138-8
- 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, PubMedID: 25819956, DOI: 10.1128/AEM.00507-15
- 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, PubMedID: 25724649, DOI: 10.1074/jbc.M114.629022
- Binyamin D Berkovits, Christine Mayr 2015. Alternative 3' UTRs act as scaffolds to regulate membrane protein localization. Nature. , PubMedID: 25896326, DOI: 10.1038/nature14321
- 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
- 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
- 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, 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, PubMedID: 25802332, DOI: 10.1074/jbc.M115.645903
- 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
- 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, PubMedID: 25173974, DOI: 10.1016/j.pep.2014.08.003
- 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, PubMedID: 25126785, DOI: 10.1016/j.cell.2014.06.047
- 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, PubMedID: 25104393, DOI: 10.1126/science.1255215
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