Pfu-DNA Polymerase
Pfu-DNA Polymerase Recombinant

Synonyms:
DNA polymerase, EC 2.7.7.7, Pfu polymerase, Pfu-DNA Polymerase.

 Introduction:
Pfu DNA polymeraseenzyme is found in the hyperthermophilic archaeonPyrococcus furiosus, where it functions in vivoto replicate the organism's DNA. In vitro, Pfu is used to swiftly amplify DNAin the Polymerase Chain Reaction, where the enzyme serves the central function of copying a new strand of DNA during each extension step. Pfu DNA polymerase has superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proof reading activity, meaning that it works its way along the DNA from the 5' endto the 3' endand corrects nucleotidemisin corporation errors. Pfu DNA polymerase-generated PCRfragments will have fewer errors than Taq-generated PCR inserts. As a result, Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq. Pfu DNA polymerase is superior for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity.

 Description:
Pfu DNA polymerase is a cloned pyrococcus furiosus DNA polymerase (Pfu) containing thermostable dUTPase that enhances PCR product yields and increases target length capability without altering DNA replication fidelity. The main inhibitor of PCR with pfu DNA polymerase is the dUTP generated from the dCTP deamination during PCR reaction. Removing dUTP from PCR reaction by thermostable dUTPase can increase the yield and length of the product.
Pfu DNA polymerase can be used to amplify complex genomic DNA targets up to 19 kb and vector targets up to 15 kb in length.

 Source:
Escherichia Coli.

  10X PCR Buffer with MgSO₄:
200mM Tris-HCl (pH 8.8 at 25°C), 100mM (NH₄)₂SO₄, 100mM KC1, 1% Triton X-100, 1mg/ml BSA, 20mM MgSO₄.

 PCR Protocol:
1. Add the following component to a PCR tube at ambient temperature or on ice: Template DNA (1-200ng): 1µl
10x PCR buffer: 5µl
10mM dNTPs: 1µl
Primers mix (10M each): 2µl
Pfu-DNA Polymerase: 1µl
Water: 40µl
Total Volume 50µl
For greater than 1kb target 1U is sufficient.
When amplifying targets greater than 3kb, 2.5U may be required.

2. Amplify using the following cycling parameters:
1x cycle: 95°C for 3minutes
30x cycles: 94°C, 30sec/58°C, 30 sec/72°C for 1 minute/1kb
1x cycle: 72°C for 7 minutes.

3. Remove 10µl from reaction and analyze by agarose gel electrophoresis.

 Applications:
1. Ideal for high-fidelity amplification.
2. 3'-5' exonuclease activity provides a low error rate.
3. One of the most thermostable DNA polymerases known.
4. Lack of extendase activity means no unwanted 3’ overhangs.
5. Optimal for blunt-end PCR cloning.
6. Optimum temperature near 75°C.
7. 95% active after 1-hour incubation at 98°C.

 References:
1. Dabrowski S. and Ahring B.K. protein Exp. Pur. 2003,31,72-78
2. Hogrefe H. et al. Proc. Natl. Acad. Sci. USA 2002,99,596-601

 Usage:
Prospec's products are furnished forLABORATORY RESEARCHUSEONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.