WITH CHARLES BOGDANSKI, ELYSE CORBIN, AND DR. MICHAEL SEHORN, DEPARTMENT OF GENETICS AND BIOCHEMISTRY
Evaluation of Pfu-Sso7d Polymerase
DNA polymerases are essential enzymes responsible for the replication and repair of DNA in living organisms. They catalyze the synthesis of a new DNA strand by adding nucleotides to a pre-existing primer strand in a sequence-specific manner, using a template strand to guide the incorporation of complementary nucleotides. DNA polymerases play crucial roles in cellular processes such as replication, recombination, and DNA repair, ensuring the accurate transmission of genetic information. The ability of these enzymes to replicate DNA was exploited in molecular biology through its use in the polymerase chain reaction (PCR), DNA sequencing, and cloning. The two primary polymerases used in PCR are Taq polymerase from the thermophilic bacterium Thermus aquaticus and Pfu polymerase from the hyperthermophilic archaeon Pyrococcus furiousus. Taq polymerase is more processive than Pfu while Pfu has proofreading capability that greatly increases its accuracy. Pfu-Sso7d is the result of fusing a DNA binding protein to the Pfu polymerase which significantly increases the processivity of Pfu. In this study, Pfu-Sso7d was expressed and purified. PCR was optimized using the Pfu-Sso7d and compared to PCR reactions mediated by Taq and Pfu polymerase. The results from this study will be used to guide our PCR-mediated site-directed mutagenesis efforts.
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