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]