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With that approach, melting curve analysis of the PCR products can distinguish the specific mutant PCR product from wild-type and other mutants. Author information Article notes Copyright and License information Disclaimer. Cell densities were determined using a hemocytometer.
The apparent increase in sensitivity seems to be achieved higj the use of a mutation-specific hybridization probe.
Phusion High-Fidelity DNA Polymerase
BoxStavanger, Norway on. Possibly, all kinds of mutations polymerasee introduced in the PNA-target, but only the most destabilizing mutants become sufficiently amplified to be observed by sequencing. Support Center Support Center.
PCR-induced errors may cause cloning errors and overestimation of genetic diversity. Our data suggest that the replication errors caused by Taq polymerase must be taken into consideration for PNA clamp PCR and for other methods based on selective PCR amplification, and that these assays can be enhanced by high-fidelity Ginnzymes polymerases.
Phusion DNA Polymerase
The DNA sequences are shown above the chromatograms, with codons 12 and 13 underlined. However, DNA polymerase errors need consideration also for other types of assays based on selective amplification of genetic variants, such as allele-specific amplification- and restriction endonuclease-mediated selective amplification-PCR.
Thus, mutation-specific probes seem to be a way to avoid the problem with polymerase-introduced errors. Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes.
Accepted Feb Design and use of a peptide nucleic acid for detection of the heteroplasmic low-frequency mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes MELAS mutation in human mitochondrial DNA.
Thus, polymerization errors seem to be a plausible explanation for the mutations we observed when analyzing wild-type template with PNA present.
This product is manufactured by New England Biolabs, Inc.
The purchase price of this product includes a limited, non-transferable license under U. We aimed to develop a PNA-clamped real-time PCR assay for sensitive detection of all possible carcinoma-associated mutations in K- ras codons 12 and 13 in lymph nodes wt K- ras from colorectal cancer patients.
Hjgh Engl J Med. Further information can be found in our Technical Resources section or at neb. However, the hybridization probe cannot distinguish a real mutation in the original template from one introduced by the polymerase during PCR.
Phusion DNA Polymerase – New England Biolabs GmbH
No other license under this polymerasse is conveyed expressly or by implication to the oolymerase by the purchase of this product. DNA polymerase fidelity and the polymerase chain reaction. Each sample was analyzed in triplicate both with and without PNA. Thus, continued effort in the development of DNA polymerase systems with higher fidelities is in the interest of several fields of molecular diagnostics.
The unique structure and characteristics of Phusion DNA Polymerase make it a superior choice for cloning. Phusion DNA Polymerase brings together a novel Pyrococcus-like enzyme with a processivity-enhancing domain and generates PCR products with accuracy and speed previously unattainable with a single enzyme, even on your most difficult templates.
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PCR-induced polymrrase artifacts and bias: There are 12 possible missense mutations in codons 12 and 13, of which several are common. Interestingly, there is evidence indicating that guanine substitutions destabilize PNA binding more than other substitutions.
Articles from The Journal of Molecular Diagnostics: Our enhanced PNA clamp assay significantly distinguished the 10 3: Please contact busdev neb. Phusion Hot Start Flex Polymerases. Extension times are indicated in minutes.
The strategy with mutation-specific hybridization probes also suffers from the drawback that multiple hybridization probes are needed to cover all possible mutations in K- ras codon 12 and codon A potentially better explanation is that multiple mutants are introduced separately in the first part of the PCR and afterward compete during the amplification. The purchase price of this product includes a limited, non-transferable license under U.