Solid phase in vitro mutagenesis using plasmid DNA template.

  • Hultman T
  • Murby M
  • Ståhl S
 et al. 
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Abstract

Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.

Author-supplied keywords

  • bacterial
  • base sequence
  • escherichia coli
  • escherichia coli genetics
  • genetic
  • genetic techniques
  • molecular sequence data
  • mutation
  • plasmids
  • polymerase chain reaction
  • templates
  • transformation

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Authors

  • T Hultman

  • M Murby

  • S Ståhl

  • E Hornes

  • M Uhlén

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