Molecular cloning and expression of Pfu DNA polymerase gene and its application in long-distance PCR

Bing Li, Cheng Gu, Jindong Zhao

Research output: Contribution to journalArticle

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Abstract

A 2.3 kb DNA fragment containing Pfu DNA polA gene was amplified by PCR from total DNA of Pyrococcus 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 expression vector pET3d-X. The Pfu polA gene was expressed in Escherichia coli BL21 (DE3). The gene product (Pfu) was purified with heat denaturation, polyethylenemine (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.

Original languageEnglish (US)
Pages (from-to)863-867
Number of pages5
JournalChinese Science Bulletin
Volume43
Issue number10
Publication statusPublished - May 1998

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Keywords

  • Expression
  • Long-distance PCR
  • Pfu enzyme

ASJC Scopus subject areas

  • General

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