Pfu DNA polymerase
Encyclopedia
Pfu DNA polymerase
is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus
, where it functions in vivo
to replicate the organism's DNA
. In vitro
, Pfu is used to quickly amplify DNA
in polymerase chain reaction
(PCR) processes, where the enzyme serves the central function of copying a new strand of DNA during each extension step.
proofreading activity, meaning that it works its way along the DNA from the 5' end to the 3' end and corrects nucleotide
-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments 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.
Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6% mutated products when amplifying 1 kb fragments using PCR. 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.
Pfu DNA polymerase is hence 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.
Polymerase Isolated from Pyrococcus Furiosus," Strategies 4:34-35 (1991) and Gene in December of that year (Gene. 1991 Dec 12;108(1):1-6). U.S. Patent 5,489,523 was granted over exonuclease-deficient Pfu in February 1996 while U.S. and (1991)Patent 5,545,552 over Pfu itself was granted in August 1996.
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus
Pyrococcus furiosus
Pyrococcus furiosus is an extremophilic species of Archaea. It can be classified as a hyperthermophile because it thrives best under extremely high temperatures—higher than those preferred of a thermophile...
, where it functions in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
to replicate the organism's DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. In vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
, Pfu is used to quickly amplify DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
in polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) processes, where the enzyme serves the central function of copying a new strand of DNA during each extension step.
Proofreading ability of
Pfu polymerase The main difference between Pfu and alternative enzymes is Pfus superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonucleaseExonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
proofreading activity, meaning that it works its way along the DNA from the 5' end to the 3' end and corrects nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments 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.
Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6% mutated products when amplifying 1 kb fragments using PCR. 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.
Pfu DNA polymerase is hence 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.
History
Scientists associated with the biotech company Stratagene, based in La Jolla, California, discovered the enzyme on the floor of the company's headquarters, and found subsequently the superiority of Pfu over Taq in 1991, since they flamed it and it still retained its tertiary structure. They published their work as Lundberg et al., "A High Fidelity Thermostable DNAPolymerase Isolated from Pyrococcus Furiosus," Strategies 4:34-35 (1991) and Gene in December of that year (Gene. 1991 Dec 12;108(1):1-6). U.S. Patent 5,489,523 was granted over exonuclease-deficient Pfu in February 1996 while U.S. and (1991)Patent 5,545,552 over Pfu itself was granted in August 1996.