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This article has been cited by fknnzymes articles in PMC. Confidence intervals were computed using the t distribution.
Thenmalarchelvi R, Yathindra N. It is likely that both of these methods will benefit from high-fidelity DNA polymerases. The finnzymes curves of a sample must be compared with the amplification curve of wild-type template to judge whether it has a mutation or not. New England Biolabs, Inc. The purchase price of this product includes a limited, non-transferable license under a patent owned by New England Biolabs, Inc. Support Center Support Center.
Phusion DNA Polymerase
Using amplification curves to detect mutations introduces another challenge. With that approach, melting curve analysis of the PCR products can distinguish the specific mutant PCR product from wild-type and other mutants. Subsequently, several studies using similar assays to detect microscopic cancer dissemination have been reported. Author information Article notes Copyright and License information Disclaimer. Open in a separate window.
Peptide nucleic acid clamp PCR: Each sample was analyzed in triplicate both with and without PNA. In contrast, our single PNA clamp PCR assay detected different mutations in codons 12 and 13 with similar efficiency results not shown. Diagnostic biochip phsion for fast and sensitive detection of Higj mutations in stool.
We found several different mutations in the PNA-binding site Figure 5confirming our hypothesis. In their discussion of possible limitations, they mention the possibility that errors introduced by Taq polymerase can lead to false-positive results when the amount of mutant template is less than 0. 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 Although the peaks corresponding to introduced mutants po,ymerase the sequencing chromatograms were quite convincing, the wild-type peaks in the same position were usually higher Figure 5A.
Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes.
Extension times are indicated in minutes. Cell densities were determined using a hemocytometer. Novel non-templated nucleotide addition reactions dnna by procaryotic and eucaryotic DNA polymerases.
Nucleotide insertion kinetics opposite abasic lesions in DNA.
The apparent increase in sensitivity seems to be achieved by the use of a mutation-specific hybridization probe. 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. If all mutant peaks correspond to polymease type of mutant PCR product, this could indicate that the main reason for exponential amplification from wild-type template is incomplete PNA-binding and not polymerase errors.
Further information can be found in our Technical Resources section or at neb.
Phusion High-Fidelity DNA Polymerase
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.
Please contact busdev neb. Selective amplification of mutant templates polyjerase PNA clamping of the wild-type template phusioon favored amplification of PCR products with polymerase-induced errors within the PNA-binding site. PCR-induced sequence artifacts and bias: However, the hybridization probe cannot distinguish a real mutation in the original template from one introduced by the polymerase during PCR.
A potentially better explanation is that multiple mutants are introduced separately in the first part of the PCR and afterward compete during the amplification. Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: Thus, polymerization errors seem to be a plausible explanation for the mutations we observed when analyzing wild-type template with PNA present.
DNA was isolated from the cell suspensions as described above. Schematic pictorial of our PNA clamp assay.
Phusion DNA Polymerase – New England Biolabs
Thus, mutation-specific probes seem to be a way to avoid the problem with polymerase-introduced errors. Phusion Hot Start Flex Polymerases. There are 12 possible missense mutations in codons 12 and 13, of which several are common.
The purchase price of this product includes a limited, non-transferable license under U. One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and ginnzymes probes. Interestingly, there is evidence indicating that guanine substitutions destabilize PNA binding more than other substitutions. HotGoldStar polymerase is a modified recombinant Taq DNA polymerase with hot-start capabilities according to the manufacturer.