''Pfu'' DNA polymerase is an enzyme found in the hyperthermophilic archaeon '' Pyrococcus furiosus'', where it functions to copy the organism's

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

during cell division (

thermostable DNA polymerase Thermostable DNA polymerases are DNA polymerases that originate from thermophiles, usually bacterial or archaeal species, and are therefore thermostable. They are used for the polymerase chain reaction and related methods for the amplification a ...

). In the laboratory setting, ''Pfu'' is used to amplify

in the

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...

(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 create t ...

. It has an

RNase H Ribonuclease H (abbreviated RNase H or RNH) is a family of non-nucleotide sequence, sequence-specific endonuclease enzymes that catalysis, catalyze the cleavage of RNA in an RNA/DNA substrate (chemistry), substrate via a hydrolysis, hydrolytic c ...

-like 3'-5' exonuclease domain, typical of B-family polymerase such as

DNA polymerase II DNA polymerase II (also known as DNA Pol II or Pol II) is a prokaryote, prokaryotic DNA-dependent DNA polymerase encoded by the PolB gene. DNA Polymerase II is an 89.9-kDa protein and is a member of the B family of DNA polymerases. It was origin ...

Proofreading ability of ''Pfu'' polymerase

''Pfu'' DNA polymerase has superior thermostability and proofreading properties compared with ''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 th ...

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-r ...

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-ri ...

, the exonuclease activity immediately removes

nucleotides Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...

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 in San Diego, California, occupying of curving coastline along the Pacific Ocean. The population reported in the 2010 census was 46,781. The climate is mild, with an average daily temperature o ...

, discovered Pfu DNA polymerase which exhibits significantly higher fidelity of replication than Taq DNA polymerase in 1991. They received patents for exonuclease-deficient ''Pfu'' and the full ''Pfu'' in 1996., Other polymerases from ''Pyrococcus'' strains such as "Deep Vent" () from strain GB-D and Pwo DNA polymerase have 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] {{Portal bar, Biology, border=no DNA replication EC 2.7.7 Polymerase chain reaction