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Thermus aquaticus DNA polymerase I mutants with altered fidelity. Click here to opt-out.

Phusion High-Fidelity DNA Polymerase

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. PCR-induced errors may cause cloning errors and overestimation of genetic diversity.

Abstract Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer hugh the monitoring of treatment at a molecular level. Interacting mutations in the O-helix. Only the introduced mutations are shown explicitly. The purchase price of this product includes a limited, non-transferable license under U. Thenmalarchelvi R, Yathindra N.

Phusion DNA Polymerase – New England Biolabs

Thus, the PNA clamping seemed to be incomplete. Further, the use of mutation-specific hybridization probes would not enhance the assay as it would be impossible to distinguish the mutations arising during the PCR from the mutations present in the sample to be analyzed. Detection of microscopic spread of tumor cells is of interest in several areas of cancer research. Common strategies to distinguish tumor cells from normal cells are based on different histological properties, specific protein and mRNA expressions, and tumor-specific mutations.


This product is manufactured by New England Biolabs, Inc.

National Center for Biotechnology InformationU. Articles from The Journal of Molecular Diagnostics: The DNA sequences are shown above the chromatograms, with codons 12 and 13 phusioon. 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 apparent increase in sensitivity seems to be achieved by the use of a mutation-specific hybridization probe. Thus, mutation-specific probes seem to be a way to avoid the problem with polymerase-introduced errors.

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. 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 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. During the next cycles, the PNA binding to the newly synthesized strands containing errors in the PNA-binding site phusoon be reduced or completely inhibited, depending on the phusioon of mutation, and exponential amplification could occur.

There are 12 possible missense mutations in codons 12 and 13, of which several are common. Extension times are indicated in minutes. Using amplification curves to detect mutations introduces another challenge.

J Hepatobiliary Fidlity Surg. Thus, continued effort in the development of DNA polymerase systems with higher fidelities is in the interest of several fields of molecular diagnostics.

In contrast, our single PNA clamp PCR assay detected different mutations in codons 12 and 13 with similar efficiency results not shown. Journal List J Mol Diagn v. Open in a separate window.


HotGoldStar polymerase is a modified recombinant Taq DNA polymerase with hot-start capabilities according to the manufacturer. Nucleotide insertion kinetics opposite abasic lesions in DNA. Sensitive detection of tumor-specific point mutations is of interest in both the early detection of cancer and the phusino of treatment at a molecular level.

Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe polmyerase the detection of rare mutations. Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: In view of our results we emphasize the possibility for false-positives hifh to polymerase-induced errors matching the probe.

N Engl J Med. Possibly, all kinds of mutations are introduced in the PNA-target, but only the most destabilizing mutants become sufficiently ppolymerase to be observed by sequencing. Interestingly, there is evidence indicating that guanine substitutions destabilize PNA binding more than other substitutions. Reliability of the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based test for epidermal growth factor receptor mutations integrated into the clinical practice for non-small cell lung cancers.

DNA polymerase fidelity and the polymerase chain reaction.

Phusion DNA Polymerase

PCR setup and analysis were performed in separate rooms to avoid contamination. DNA was isolated from the cell suspensions as described above. Platinum Taq is a recombinant Thermus aquaticus Taq DNA polymerase with a thermolabile inhibitor to achieve hot-start capabilities according to the manufacturer.