Molecular cloning and expression of Pfu DNA polymerase gene and its application in long-distance PCR
A 2.3 kb DNA fragment containing Pfu DNA polA gene was amplified by PCR from total DNA ofPyrococcus furiosus and cloned into a pGEM-T vector. The recombinant clone pT-pfu was digested with Nco I and Xho I and the fragment was inserted into an expresion vector pET3d-X. The Pfu polA gene was expressed inEscherichia coli BL21 (DE3). The gene product (Pfu) was purified with heat denaturation, polyethyleneminc (PEI) precipitation and Bio-rex 70 ion-exchange chromatography. The recombinant Pfu was verified by Protein N-terminal sequencing. With the recombinant Pfu. large λ DNA fragments were successfully amplified in long-distance PCR.
KeywordsPfu enzyme expression long-distance PCR
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