Functional Characterization of Pol-dUT Fusion Enzymes from Pyrococcus Furiosus

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Although polymerase chain reaction is a powerful technique, often polymerizing errors can jeopardize the whole experiment especially when to study a cloned gene product. Long PCR with conventional Taq DNA polymerases is one such example, where conversion of dCTP to dUTP can increase the error frequency and dUTP incorporation as well. However, using proofreading polymerases for long PCR, the polymerization rate slows down significantly due to incorporation of dUTP during synthesis followed proofreading. To alleviate this problem, now a day we use dUTPase enzyme in the PCR. To have similar effect, our laboratory has designed two fusion enzyme constructs of both a proofreading polymerase and a dUTPase from Pyrococcus furiosus. We are reporting here that both these fusion constructs could remove dUTP from PCR reactions and able to amplify. This project particularly entails successful cloning, expression and purification of a dUTPase enzyme followed by its functional characterization in parallel with the above mentioned fusion enzymes.

Pfu polymerase, dUTPase