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Genetic alterations during colorectal-tumor development. 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.
Information on trademarks can be found here. Diagnostic biochip array for fast and sensitive detection of K-ras mutations in stool. The amplification curves of a sample must be compared with the amplification curve of wild-type template to judge whether it has a mutation or not. This article has been cited by other articles in PMC. Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic polymersse using PNA-mediated PCR clamping and hybridisation probes.
Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level. Published by Elsevier Inc.
Cell densities were determined using a hemocytometer. Wymondham, Horizon Bioscience To reduce the frequency of polymerase-induced errors, we developed a PNA clamp PCR assay for the detection of mutations in codons 12 and 13 of the K- ras gene based on a high-fidelity DNA polymerase. Interacting mutations in the O-helix. Common strategies to distinguish tumor cells from normal cells are based on different histological properties, specific protein and mRNA expressions, and tumor-specific mutations.
Thenmalarchelvi R, Yathindra N. Confidence intervals were computed using the t distribution.
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The purchase price of this product includes a limited, non-transferable license under U. Fkdelity contrast, our single PNA clamp PCR assay detected different mutations in codons 12 and 13 with similar efficiency results not shown. Polymrase potentially better explanation is that multiple mutants are introduced separately in the first phusipn of the PCR and afterward compete during the amplification. Nucleotide insertion kinetics opposite abasic lesions in DNA.
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 Abstract Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the monitoring of treatment at a molecular level.
Although the peaks corresponding to introduced mutants in the sequencing chromatograms were quite convincing, the wild-type peaks in the same position were usually higher Figure 5A. This product is manufactured by New England Biolabs, Inc.
Interestingly, there is evidence indicating that guanine substitutions destabilize PNA binding more than other substitutions. The identity of the PCR products was initially also confirmed by sequencing. Possibly, all kinds of mutations are introduced in the PNA-target, but only the most destabilizing mutants become sufficiently amplified to be observed by sequencing. One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes.
Schematic pictorial of our PNA clamp assay. finnzyems
Phusion DNA Polymerase
The unique structure and characteristics of Phusion DNA Polymerase make it a superior choice for cloning. To explore the sensitivity of our assay in a cellular context, we analyzed dilution series finnzyems 10 210 310 4and 10 5 LST phjsion in 10 7 normal lymphocytes. The colon carcinoma cell line LST was cultured as described above, grown to near confluence, and trypsinated as suggested by the European Collection of Cell Cultures.
N Engl J Med. No other license under this patent is conveyed expressly or by implication to the purchaser by the purchase of this product. 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.
The purity of all PCR products was monitored by melting curve analysis.