Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase, N.Bpu10 I, and exonuclease III

Biotechnol Lett. 2003 Nov;25(22):1913-6. doi: 10.1023/b:bile.0000003981.60247.48.

Abstract

A method to assemble linear expression elements for rapid gene expression is described. Primers containing target specific sequences and N.Bpu10 I nickase recognition sites were used to amplify promoter, open reading frame and terminator fragments. Amplified fragments were treated with N.Bpu10 I nickase and exonuclease III to generate overhangs for directional ligation. These fragments were ligated and further amplified with element-specific primers. The amplified DNA was transfected into mammalian cells for gene expression.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cattle
  • Cricetinae
  • Cricetulus
  • DNA / chemistry
  • DNA / metabolism
  • DNA Fragmentation / genetics*
  • DNA Primers / chemistry
  • Deoxyribonuclease I / chemistry*
  • Deoxyribonucleases, Type II Site-Specific / chemistry*
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Exodeoxyribonucleases / chemistry*
  • Feasibility Studies
  • Gene Expression Regulation / genetics*
  • Growth Hormone / biosynthesis*
  • Growth Hormone / genetics
  • Polymerase Chain Reaction / methods
  • Promoter Regions, Genetic / genetics
  • Protein Engineering / methods*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Terminator Regions, Genetic
  • Transfection / methods*

Substances

  • DNA Primers
  • Recombinant Proteins
  • Growth Hormone
  • DNA
  • Exodeoxyribonucleases
  • exodeoxyribonuclease III
  • endodeoxyribonuclease Bpu10I
  • Deoxyribonuclease I
  • Deoxyribonucleases, Type II Site-Specific