Pfu DNA Polymerase
Description
Product Specs
0.2µl Pfu-DNA Polymerase.
4µl 2.5mM dNTPs.
5µl 10x buffer with MgSO4.
1µl Primers mix (10µM each).
1µl Template.
38µl ddH2O.
Amplify using the following cycling parameters:
Heat Soak: 1 cycle at 94°C/4 min.
Denaturation: 30 cycles at 94°C/30 sec.
Annealing: 30 cycles at 55°C /30 sec.
Extension: 30 cycles at 72°C /90 sec.
Final: 1 cycle at 72°C /5 min.
Product Science Overview
Introduction
Pfu-DNA Polymerase is a DNA polymerase enzyme isolated from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme is renowned for its exceptional thermostability and high fidelity, making it a popular choice for various molecular biology applications, particularly the polymerase chain reaction (PCR). The recombinant form of Pfu-DNA Polymerase is produced by cloning the gene encoding the enzyme into Escherichia coli and expressing it in these bacterial cells.
Discovery and Significance
The discovery of Pfu-DNA Polymerase dates back to the early 1990s when scientists identified its superior fidelity compared to the widely used Taq DNA polymerase . The enzyme’s high fidelity is attributed to its 3’ to 5’ exonuclease activity, which provides a proofreading function, allowing it to correct nucleotide incorporation errors during DNA synthesis . This proofreading ability results in a significantly lower error rate, making Pfu-DNA Polymerase an ideal choice for applications requiring high accuracy, such as cloning and sequencing.
Preparation and Expression
The recombinant Pfu-DNA Polymerase is prepared by inserting the gene encoding the enzyme into a plasmid vector, which is then introduced into E. coli cells. The expression of the enzyme is induced, and the cells are harvested and lysed to release the enzyme. The enzyme is then purified using techniques such as affinity chromatography . The recombinant form retains the high fidelity and thermostability of the native enzyme, making it suitable for various research applications.
Applications
Pfu-DNA Polymerase is widely used in PCR due to its high fidelity and ability to amplify DNA with minimal errors. It is particularly useful in applications where accuracy is critical, such as:
- Cloning: Generating PCR products for cloning into vectors.
- Sequencing: Amplifying DNA for sequencing to ensure accurate results.
- Site-Directed Mutagenesis: Introducing specific mutations into DNA sequences.
- Blunt-End Cloning: Producing PCR products with blunt ends for cloning.
Conclusion
Pfu-DNA Polymerase recombinant is a valuable tool in molecular biology, offering high fidelity and thermostability for various applications. Its ability to correct errors during DNA synthesis makes it an essential enzyme for researchers requiring precise DNA amplification.
