What is pfu enzyme？

一、Source

1. Microbial Characteristics
   • Pyrococcus furiosus is an archaeon living in high – temperature environments, usually found in high – temperature waters near deep – sea hydrothermal vents or volcanic craters. This microorganism can survive and reproduce in an environment close to 100 °C, and various enzymes inside its cells have high thermal stability to adapt to the extreme living environment.
2. Enzyme Extraction
   • The PFU enzyme is obtained through culturing Pyrococcus furiosus, cell disruption, and a series of complex biochemical separation and purification techniques. The extraction process needs to be carried out under strictly controlled conditions to ensure the activity and purity of the enzyme.

二、Enzyme Characteristics

1. Thermal Stability
   • The PFU enzyme has excellent thermal stability and can maintain its activity at high temperatures. Its optimal reaction temperature is usually around 70 – 75 °C. Within this temperature range, the enzyme can efficiently catalyze DNA synthesis reactions. Even after incubation at a high temperature of 95 °C for a period of time, it can still retain a considerable part of its activity, which gives it a great advantage in DNA manipulation experiments requiring high – temperature reaction conditions.
2. High Fidelity
   • It has a high degree of fidelity and can accurately synthesize a new DNA strand according to the template strand during DNA replication, introducing very few incorrect bases. This high – fidelity characteristic is due to the fact that the PFU enzyme itself has 3′ – 5′ exonuclease activity, which can proofread the newly synthesized DNA strand and promptly excise mismatched bases to ensure the accuracy of DNA replication.
3. Catalytic Activity
   • The PFU enzyme can use single – stranded or double – stranded DNA as a template to catalyze dNTPs (deoxynucleoside triphosphates) to synthesize new DNA strands. Under appropriate reaction buffer and substrate concentration conditions, it can rapidly extend the DNA strand to complete operations such as DNA replication and amplification.

三、Applications

1. Polymerase Chain Reaction (PCR)
   • In conventional PCR experiments, the PFU enzyme can be used for DNA amplification requiring high fidelity. For example, when cloning genes, if a DNA polymerase with low fidelity is used, mutations may be introduced during the amplification process, resulting in inaccurate cloned genes. The PFU enzyme can effectively avoid this situation, ensuring that the amplified DNA fragments are highly consistent with the original template, which is beneficial for subsequent gene function research and expression vector construction.
2. DNA Sequencing
   • In DNA sequencing reactions, the PFU enzyme can accurately synthesize DNA strands complementary to the template, which is crucial for obtaining accurate DNA sequence information. It can reduce sequencing errors caused by base mismatches and improve the reliability and accuracy of sequencing results, having important applications in genome sequencing, gene variation detection, and other fields.
3. Site – Directed Mutagenesis Research
   • In gene site – directed mutagenesis experiments, the PFU enzyme can be used to construct mutants. Due to its high fidelity and precise DNA – synthesizing ability, researchers can more accurately introduce expected mutations at specific positions while avoiding unnecessary random mutations, facilitating the study of the effects of specific gene mutations on their functions.