Two-Step Red-Mediated Recombination for versatile High-Efficiency Markerless DNA Manipulation in Escherichia Coli

Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis…

• MA
  Morris Animal Foundation

  Award: D03EQ-74
• NI
  National Institutes of Health

  Award: AI060843-02