preliminary expression of high fidelity pfu dna polymerase encoding fragment
نویسندگان
چکیده
منابع مشابه
Crystal structure of Pfu, the high fidelity DNA polymerase from Pyrococcus furiosus.
We have determined a 2.6A resolution crystal structure of Pfu DNA polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus. Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The inter...
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The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated...
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We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a sin...
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Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequen...
متن کاملConstruction, Expression and Preliminary Immunological Evaluation of a DNA Plasmid Encoding the GRA2 Protein of Toxoplasma gondii
Toxoplasmosis is a worldwide infection which is commonly asymptomatic but can cause serious medical problems in immunocompromised individuals and fetus. The infection also causes considerable economic loss because of abortion in livestock, mostly in sheep and goats. DNA vaccination may be a powerful approach against intracellular parasites such as Toxoplasma gondii. The goal of this study was t...
متن کاملDNA Polymerase Selectivity: Sugar Interactions Monitored with High-Fidelity Nucleotides
The essential prerequisite of any organism is to keep its genome intact and to accurately duplicate it before cell division. All DNA synthesis required for DNA repair, recombination, and replication depends on the ability of DNA polymerases to recognize the template and correctly insert the complementary nucleotide. In a current model, fidelity is achieved by the ability of DNA polymerases to e...
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عنوان ژورنال:
research in pharmaceutical sciencesجلد ۷، شماره ۵، صفحات ۰-۰
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