''Pfu'' DNA polymerase is an enzyme found in the hyperthermophilic
archaeon
Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebact ...
''
Pyrococcus furiosus
''Pyrococcus furiosus'' is a heterotrophic, strictly anaerobic, extremophilic, model species of archaea. It is classified as a hyperthermophile because it thrives best under extremely high temperatures, and is notable for having an optimum gr ...
'', where it functions to copy the organism's
DNA during cell division. In the laboratory setting, ''Pfu'' is used to amplify
DNA in the
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
(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
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to crea ...
. It has an
RNase H
Ribonuclease H (abbreviated RNase H or RNH) is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/ DNA substrate via a hydrolytic mechanism. Members of the RNase H family can be found in nearly ...
-like 3'-5' exonuclease domain, typical of B-family polymerase such as
DNA polymerase II.
Proofreading ability of ''Pfu'' polymerase
''Pfu'' DNA polymerase has superior thermostability and proofreading properties compared to
''Taq'' DNA polymerase. Unlike ''Taq'' DNA polymerase, ''Pfu'' DNA polymerase possesses 3' to 5'
exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is ...
proofreading activity, meaning that as the DNA is assembled from the
5' end
Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar- ...
to
3' end
Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-r ...
, the exonuclease activity immediately removes
nucleotides
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules with ...
misincorporated at the 3' end of the growing DNA strand. Consequently, ''Pfu'' DNA polymerase-generated PCR fragments will have fewer errors than ''Taq''-generated PCR inserts.
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 to ''Taq'' DNA polymerase 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 led by Eric Mathur at the biotech company Stratagene, based in La Jolla, California
La Jolla ( , ) is a hilly, seaside neighborhood within the city of San Diego, California, United States, occupying of curving coastline along the Pacific Ocean. The population reported in the 2010 census was 46,781.
La Jolla is surrounded on ...
, discovered Pfu DNA polymerase which exhibits significantly higher fidelity of replication than Taq DNA polymerase in 1991.[, ]
Other polymerases from ''Pyrococcus'' strains such as "Deep Vent" () from strain GB-D and Pwo DNA polymerase has also seen use.
References
External links
Stratagene's ''Pfu'' U.S. Patents
*[https://web.archive.org/web/20120309153834/http://www.patentlens.net/patentlens/patents.html?patnums=US_5545552&language=en&query=(US_5545552%20in%20publication_number)&stemming=true&returnTo=patentnumber.html%3Fquery=&stemming=true&patentNumber=5%2C545%2C552&collections=US_B,EP_B,AU_B,US_A,WO_A,AU_A&language=en Patent 5,545,552]
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DNA replication
EC 2.7.7
Polymerase chain reaction