Pfu DNA polymerase


Pfu DNA polymerase

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 ability of Pfu polymerase

The main difference between Pfu and alternative enzymes is Pfu's superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease 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 1kb 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 superiority of Pfu over Taq in 1991. They published their work as Bergseid 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. [http://www.bios.net/patentlens/simple.cgi?form_num=5&mode=show&version=&options=&form_bools=AND&collection=WO_A&collection=EP_B&collection_all=US_A_all&collection_all=US_B_all&field_form_0=in%20fulltext&bool_form_0=&query_0=&field_form_1=in%20title&bool_form_1=AND&query_1=&field_form_2=in%20abstract&bool_form_2=AND&query_2=&field_form_3=in%20inventor&bool_form_3=AND&query_3=&field_form_4=in%20applicant&bool_form_4=AND&query_4=&field_form_5=Patent_num&bool_form_5=AND&coll_type_5=US&query_5=5%2C489%2C523&action=show&history=--%20Search%20history%20--&show=patent&sort=patent&page_length=10&stemming=yes&relevance_bool=&rel_tfidf=5&rel_tf_ti=3&rel_tf_ab=2&rel_tf_cl=4&rel_tf_ft=1&rel_prox=5&rel_pr_ti=3&rel_pr_ab=2&rel_pr_cl=4&rel_pr_ft=1&rel_bool=0&rel_bl_ti=3&rel_bl_ab=2&rel_bl_cl=4&rel_bl_ft=1&rel_bool_sel=1&sort_order=ascending&query=US%205489523&file=US_5489523&.cgifields=show&.cgifields=collection_all&.cgifields=collection#list Patent 5,489,523] was granted over exonuclease-deficient Pfu in February 1996 while U.S. and (1991) [http://www.bios.net/patentlens/simple.cgi?form_num=5&mode=show&version=&options=&form_bools=AND&collection=WO_A&collection=EP_B&collection_all=US_A_all&collection_all=US_B_all&field_form_0=in%20fulltext&bool_form_0=&query_0=&field_form_1=in%20title&bool_form_1=AND&query_1=&field_form_2=in%20abstract&bool_form_2=AND&query_2=&field_form_3=in%20inventor&bool_form_3=AND&query_3=&field_form_4=in%20applicant&bool_form_4=AND&query_4=&field_form_5=Patent_num&bool_form_5=AND&coll_type_5=US&query_5=5%2C545%2C552&action=show&history=--%20Search%20history%20--&show=patent&sort=patent&page_length=10&stemming=yes&relevance_bool=&rel_tfidf=5&rel_tf_ti=3&rel_tf_ab=2&rel_tf_cl=4&rel_tf_ft=1&rel_prox=5&rel_pr_ti=3&rel_pr_ab=2&rel_pr_cl=4&rel_pr_ft=1&rel_bool=0&rel_bl_ti=3&rel_bl_ab=2&rel_bl_cl=4&rel_bl_ft=1&rel_bool_sel=1&sort_order=ascending&query=US%205545552&file=US_5545552&.cgifields=show&.cgifields=collection_all&.cgifields=collection#list Patent 5,545,552] over Pfu itself was granted in August 1996.

External links

* [http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2Fsearch-adv.htm&r=0&f=S&l=50&d=PTXT&RS=%28ABST%2F%22Pyrococcus+furiosus%22+AND+ABST%2Fpolymerase%29&Refine=Refine+Search&Query=abst%2F%22Pyrococcus+furiosus%22+and+abst%2Fpolymerase+and+an%2Fstratagene Stratagene's Pfu U.S. Patents]
* [http://www.stratagene.com/products/displayProduct.aspx?pid=416 Pfu information from the Stratagene web site]
* [http://www.bios.net/patentlens/simple.cgi?form_num=5&mode=show&version=&options=&form_bools=AND&collection=WO_A&collection=EP_B&collection_all=US_A_all&collection_all=US_B_all&field_form_0=in%20fulltext&bool_form_0=&query_0=&field_form_1=in%20title&bool_form_1=AND&query_1=&field_form_2=in%20abstract&bool_form_2=AND&query_2=&field_form_3=in%20inventor&bool_form_3=AND&query_3=&field_form_4=in%20applicant&bool_form_4=AND&query_4=&field_form_5=Patent_num&bool_form_5=AND&coll_type_5=US&query_5=5%2C489%2C523&action=show&history=--%20Search%20history%20--&show=patent&sort=patent&page_length=10&stemming=yes&relevance_bool=&rel_tfidf=5&rel_tf_ti=3&rel_tf_ab=2&rel_tf_cl=4&rel_tf_ft=1&rel_prox=5&rel_pr_ti=3&rel_pr_ab=2&rel_pr_cl=4&rel_pr_ft=1&rel_bool=0&rel_bl_ti=3&rel_bl_ab=2&rel_bl_cl=4&rel_bl_ft=1&rel_bool_sel=1&sort_order=ascending&query=US%205489523&file=US_5489523&.cgifields=show&.cgifields=collection_all&.cgifields=collection#list Patent 5,489,523]
* [http://www.bios.net/patentlens/simple.cgi?form_num=5&mode=show&version=&options=&form_bools=AND&collection=WO_A&collection=EP_B&collection_all=US_A_all&collection_all=US_B_all&field_form_0=in%20fulltext&bool_form_0=&query_0=&field_form_1=in%20title&bool_form_1=AND&query_1=&field_form_2=in%20abstract&bool_form_2=AND&query_2=&field_form_3=in%20inventor&bool_form_3=AND&query_3=&field_form_4=in%20applicant&bool_form_4=AND&query_4=&field_form_5=Patent_num&bool_form_5=AND&coll_type_5=US&query_5=5%2C545%2C552&action=show&history=--%20Search%20history%20--&show=patent&sort=patent&page_length=10&stemming=yes&relevance_bool=&rel_tfidf=5&rel_tf_ti=3&rel_tf_ab=2&rel_tf_cl=4&rel_tf_ft=1&rel_prox=5&rel_pr_ti=3&rel_pr_ab=2&rel_pr_cl=4&rel_pr_ft=1&rel_bool=0&rel_bl_ti=3&rel_bl_ab=2&rel_bl_cl=4&rel_bl_ft=1&rel_bool_sel=1&sort_order=ascending&query=US%205545552&file=US_5545552&.cgifields=show&.cgifields=collection_all&.cgifields=collection#list Patent 5,545,552]


Wikimedia Foundation. 2010.

Look at other dictionaries:

  • Pfu-ДНК-полимераза — * Pfu ДНК полімераза * Pfu DNA polymerase высокотермостабильная и гиперактивная мономерная ДНК полимераза с 51 31 полимеразной и 31 51 эндонуклеазной активностью. Изолирована из сверхтермофильной морской архебактерии Pyrococcus furiosis (Pfu).… …   Генетика. Энциклопедический словарь

  • Polymerase chain reaction optimization — The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure. Contents 1… …   Wikipedia

  • Pfu-Polymerase — DNA Polymerase (Pyrococcus furiosus) Vorhandene Strukturdaten …   Deutsch Wikipedia

  • Polymerase Chain Reaction — Die Polymerase Kettenreaktion (englisch Polymerase Chain Reaction, PCR) ist eine Methode, um die Erbsubstanz DNA in vitro zu vervielfältigen. Dazu wird ein Enzym verwendet, die DNA Polymerase. Der Begriff Kettenreaktion beschreibt in diesem… …   Deutsch Wikipedia

  • Polymerase-Kettenreaktion — Die Polymerase Kettenreaktion (englisch Polymerase Chain Reaction, PCR) ist eine Methode, um die Erbsubstanz DNA in vitro zu vervielfältigen. Dazu wird ein Enzym verwendet, die DNA Polymerase. Der Begriff „Kettenreaktion” beschreibt in… …   Deutsch Wikipedia

  • Polymerase Chain Reaction — Réaction en chaîne par polymérase Pour les articles homonymes, voir PCR. Diagramme des 4 premiers cycles de la PCR La réaction en chaîne par polymérase (PCR est l abréviation an …   Wikipédia en Français

  • Taq polymerase — is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolatedcite journal|author=Chien A, Edgar DB, Trela JM|year= 1976|title=Deoxyribonucleic acid polymerase from the extreme… …   Wikipedia

  • Pwo-Polymerase — DNA Polymerase (Pyrococcus woesei) Masse/Länge Primärstruktur 779 Aminosäuren …   Deutsch Wikipedia

  • Taq-Polymerase — I Vorhandene Strukturdaten: s. UniProt Masse/Länge …   Deutsch Wikipedia

  • Reaction En Chaine Par Polymerase — Réaction en chaîne par polymérase Pour les articles homonymes, voir PCR. Diagramme des 4 premiers cycles de la PCR La réaction en chaîne par polymérase (PCR est l abréviation an …   Wikipédia en Français