Common questions

Is Pfu polymerase is thermostable?

Is Pfu polymerase is thermostable?

Pfu DNA Polymerase is a high-fidelity, thermostable enzyme of approximately 90kDa isolated from Pyrococcus furiosus.

What is thermostable polymerase?

A thermostable DNA polymerase is used in repeated cycles of primer annealing, DNA synthesis and dissociation of duplex DNA to serve as new templates. The theoretical amplification of template DNA, assuming reagents are not limiting and the enzyme maintains full activity, is 2n where n is the number of cycles.

Is Pfu polymerase 3 thermostable?

3′-5′ exonuclease activity provides a low error rate. 3. One of the most thermostable DNA polymerases known.

Is PFU a high fidelity polymerase?

High-Fidelity PCR. Pfu DNA Polymerase is a high-fidelity, thermostable enzyme of approximately 90kDa isolated from Pyrococcus furiosus. The enzyme replicates DNA at 75°C, catalyzing the polymerization of nucleotides in the 5´→3´ direction in the presence of magnesium.

Is E coli DNA polymerase thermostable?

This thermostable DNA polymerase or “Taq” enzyme was found to possess similar properties to E. coli DNA dependant DNA polymerase I, with strong homology being found at the amino acid level so: the 3′-OH nucleotide addition site, the dNTP/DNA binding sites, and the 5′-3′ exonuclease sites of the two enzymes (Fig. 7.1).

Is PFU a high-fidelity polymerase?

What is the reaction buffer for Pfu DNA polymerase?

Reaction Buffer In Pfu DNA polymerase-based PCR, the reaction buffers for native and cloned Pfu DNA polymerases are formulated for optimal PCR yield and fidelity.6 Use the 10× reaction buffer provided with each polymerase for all PCR applications.

How is PFU different from Taq DNA polymerase?

Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3′ to 5′ exonuclease proofreading activity, meaning that as the DNA is assembled from the 5′ end to 3′ end, the exonuclease activity immediately removes nucleotides misincorporated at the 3′ end of the growing DNA strand.

How is PFU used in the PCR process?

In the laboratory setting, Pfu is used to amplify DNA in the polymerase chain reaction (PCR), where the enzyme serves the central function of copying a new strand of DNA during each extension step. It is a family B DNA polymerase. It has an RNase H-like 3′-5′ exonuclease domain, typical of B-family polymerase such as DNA polymerase II.

How long does PFU take to amplify 1kb of DNA?

However, Pfu is slower and typically requires 1–2 minutes per cycle to amplify 1kb of DNA at 72 °C. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products.

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