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 PC R from total DNA of Pyrococcus furiosus and cloned into a pGEM-T vecto r. The recombinant clone pT-pfu was digested with Nco I and Xho I and the fragment was inserted into an expression vector pET3d-X. The Pfu p olA gene was expressed in Esherichia coli BL21 (DE3). The gene product (Pfu) was purified with heat denaturation, polyethylenemine (PEI) pre cipitation and Bio-rex 70 ion-exchange chromatography. The recombinant Pfu was verified by protein N-terminal sequencing. With the recombina nt Pfu, large lambda DNA fragments were successfully amplified in long -distance PCR.