A Modified Inverse PCR Procedure for Insertion, Deletion, or Replacement of a DNA Fragment in a Target Sequence and Its Application in the Ligand Inte

Functional analysis of a protein of interest, by generation of functional alterations in a target protein, often requires the performance of site-directed mutagenesis within the gene sequence. These manipulations are usually performed using “cut and paste” techniques, combined with PCR. Here we describe a simple and general procedure to specifically insert a DNA fragment into any site within a given DNA sequence. We demonstrate this insertional mutagenesis by describing the insertion of a tetracysteine (4C) hexapeptide-encoding sequence into the coding sequence of the antibiotic hydrolyzing enzyme TEM-1 β-lactamase. This procedure could also be applied to insert different DNA sequences or to replace, or delete, existing fragments in a given gene. We have recently used this procedure to develop a general method (ligand interaction scan – LIScan) to generate ligand-regulated proteins.