Phusion Hot Start II High-Fidelity DNA Polymerase


The most accurate hot start DNA polymerase on the market featuring extreme specificity and improved performance.
  
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Thermo Scientific Phusion Hot Start II High Fidelity DNA Polymerase is the most accurate hot start DNA polymerase on the market. It combines the Phusion DNA Polymerase and a reversibly bound, specific Affibody ligand. Affibody inhibits the DNA polymerase activity at room temperature and thus prevents the amplification of nonspecific products.

The Affibody ligand also inhibits the 3´→5´ exonuclease activity of the polymerase, preventing degradation of primers and template DNA during reaction set up. At polymerization temperatures, the ligand is released, rendering the polymerase fully active. Phusion Hot Start II DNA Polymerase does not require a separate activation step in the PCR protocol as it is immediately reactivated at high temperatures.

New Phusion Green Hot Start II High-Fidelity DNA Polymerase is a combination of Phusion Hot Start II DNA Polymerase and 5X Phusion Green Buffers. The buffers include a density reagent and two tracking dyes for direct loading of PCR products on a gel. The colored buffer does not interfere with excelent performance of Phusion Hot Start II DNA Polymerase and is compatible with downstream applications such as DNA sequencing, ligation and restriction digestion.

Phusion PolymerasesDID YOU KNOW?

The optimal annealing temperature for Phusion DNA Polymerases may differ significantly from that of Taq-based polymerases.

For optimal results start by accurately calculating your Tm with our Tm calculator.

Highlights

  • Reaction set up can be done at room temperature
  • Reduces non-specific amplification
  • Prevents primer degradation during set up
  • Zero-time reactivation due to unique hot start technology
  • Extreme fidelity (52X Taq)
  • Speed of the polymerase allows short extension times
  • Robust reactions, minimal optimization needed
  • Increased product yields with minimal enzyme amounts
  • Direct loading on gel with Green Buffer

Applications

  • High-fidelity PCR
  • High throughput
  • Difficult (GC-rich) templates
  • Template generation for sequencing
  • Multiplex PCR
  • Long-range PCR
  • Cloning
  • Mutagenesis†
  • Microarray

Phusion Site-Directed Mutagenesis Kit also available.

Includes

Phusion Hot Start II High-Fidelity DNA Polymerase

  • Phusion Hot Start II High-Fidelity DNA Polymerase (2 U/µL)
  • 5X Phusion HF Buffer
  • 5X Phusion GC Buffer
  • DMSO
  • 50 mM MgCl2 solution

Phusion Green Hot Start II High-Fidelity DNA Polymerase

  • Phusion Hot Start II High-Fidelity DNA Polymerase (2 U/µL)
  • 5X Phusion Green HF Buffer
  • 5X Phusion Green GC Buffer
  • DMSO
  • 50 mM MgCl2 solution

Both Phusion and Phusion Green Buffers provide 1.5 mM MgCl2 in the final 1X concentration.

  
HazardousNo
Shelf Life24 Months
Shipping ConditionDry Ice
Storage buffer20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, stabilizers, 200 µg/mL BSA, and 50% glycerol.
Storage Condition-20 C
Gel image comparing Phusion Hot Start II DNA Polymerase to other suppliers' polymerases

Phusion Hot Start II DNA Polymerase provides extreme specificity and abundant yields.

Gel image comparing Phusion Hot Start II DNA Polymerase to other suppliers' polymerases

Five proofreading DNA polymerases from major suppliers were used to amplify 1.7 to 2.3 kb amplicons from human genomic DNA. All amplifications were performed in accordance with manufacturers instructions. Phusion Hot Start II DNA Polymerase provided high yields of specific products whereas all other enzymes delivered lower or no yields, some of them also amplifying non-specific products.


References

Citations

Polymerase structure

  1. Y. Wang et al., A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research. 32, 1197-1207 (2004).

  2. Wikman et al., Selection and characterization of HER2/neu-binding affibody ligands. Protein Eng Des Sel. 17, 455-462 (2004).

  3. A. Guagliardi et al., The Sso7d protein of Sulfolobus solfataricus:in vitro relationship among different activities. Archaea. 1, 87-93 (2002).

  4. Nord et al., Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature Biotechnol. 15, 772-777 (1997).

Fidelity

  1. B. Gilje et al., High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations. J Mol Diagn. 10, 325-331 (2008).

  2. L. Meng et al., BEAMing up for detection and quantification of rare sequence variants. Nature Methods. 3, 95-97 (2006).

  3. B. Frey, B. Suppmann, Demonstration of the Expend PCR Systems greater fidelity and higher yields with a lacI-based fidelity assay. Biochemica. 2, 34-35 (1995).

PCR applications

  1. S. Qiu et al., Nucleotide diversity in Silene latifolia autosomal and sex-linked genes. Proc Biol Sci. 277(1698), 3283-3290 (7 November 2010).

  2. D. G. Gibson et al., Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. Science. 319, 1215-1220 (2008). [On Science website, see the Supporting Online Material for complete Materials and Methods section]

  3. Y. Zheng, R. J. Roberts, Selection of restriction endonucleases using artificial cells. Nucleic Acids Research. 35, e83 doi: 10.1093/nar/gkm410 (2007).

  4. J. Harholt et al., ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in biosynthesis of pectic arabinan in Arabidopsis. Plant Physiol. 140, 49-58 (2006).

  5. J. P. Balhoff, G. A. Wray, Evolutionary analysis of the well characterized endo16 promoter reveals substantial variation within functional sites. Proc Natl Acad Sci USA  102, 8591–8596 (2005).

  6. F. Delbos et al., Contribution of DNA polymerase eta to immunoglobulin gene hypermutation in the mouse. J Exp Med 201, 1191–1196 (2005).

  7. B. Dummitt et al., Yeast glutamine-fructose-6-phosphate aminotransferase (Gfa1) requires methionine aminopeptidase activity for proper function. J Biol Chem 280, 14356–14360 (2005).

  8. C. S. Fernandez et al., Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost. J Virol. 79, 5721–5731 (2005).

  9. S. Fiorucci et al., A FXR-SHP regulatory cascade modulates TIMP-1 and MMPs expression in HSCs and promotes resolution of liver fibrosis. J Pharmacol Exp Ther 314, 584-595 (2005).

  10. L. Fredriksson et al., Structural requirements for activation of latent platelet-derived growth factor-CC by tissue plasminogen activator. J Biol Chem. 280, 26856–26862 (2005).

  11. M. Gauster et al., Endothelial lipase is inactivated upon cleavage by the members of the proprotein convertase family. J Lipid Res 46, 977–987 (2005).

  12. R. A. Hoskins et al., Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP). Nucleic Acids Res. 33, e185 (2005).

  13. N. Ivashikina et al., AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive. J Gen Physiol. 125, 483–492 (2005).

  14. S. M. Julio, P. A. Cotter, Characterization of the filamentous hemagglutinin-like protein FhaS in Bordetella bronchiseptica. Infect Immun. 73, 4960-4971 (2005).

  15. G. I. Karras et al., The macro domain is an ADP-ribose binding module. EMBO J 24, 1911–1920 (2005).

  16. K. Moon et al., Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT. J Bacteriol. 187, 5732-5741 (2005).

  17. T. Nawy et al., Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell. 17, 1908–1925 (2005).

  18. Y. Noutoshi et al., ALBINO AND PALE GREEN 10 encodes BBMII isomerase involved in the histidine biosynthesis in Arabidopsis thaliana. Plant Cell Physiol. 46, 1165-1172 (2005).

  19. G. Rizzo et al., The methyl transferase PRMT1 functions as co-activator of farnesoid X receptor (FXR)/9-cis retinoid X receptor and regulates transcription of FXR responsive genes. Mol Pharmacol. 68, 551-558 (2005).

  20. M. V. Rockman et al., Ancient and recent positive selection transformed opioid cis-regulation in humans. PLoS Biol. 3, e387 (2005).

  21. E. Rosonina et al., Role for PSF in mediating transcriptional activator-dependent stimulation of pre-mRNA processing in vivo. Mol Cell Biol. 25, 6734-6746 (2005).

  22. P. O. Widlund, T. N. Davis, A high-efficiency method to replace essential genes with mutant alleles in yeast. Yeast. 22, 769-774 (2005).

  23. R. Zhao et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem. 280, 22788-22792 (2005).

  24. J. E. Collins et al., A genome annotation-driven approach to cloning the human ORFeome. Genome Biol. 5, R84 (2004).

  25. M. W. Davis et al., A conserved metalloprotease mediates ecdysis in Caenorhabditis elegans. Development. 131, 6001–6008 (2004).

  26. A. Ludwig et al., Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains. J Bacteriol. 186, 5311-5320 (2004).

454 sequencing

  1. J. Y. Zhu et al., Identification of Novel Epstein-Barr Virus MicroRNA Genes from Nasopharyngeal Carcinomas. J Virol. 83, 3333-3341 (2009).