A modified inverse PCR procedure for insertion, deletion, or replacement of a DNA fragment in a target sequence and its application in the ligand interaction scan method for generation of ligand-regulated proteins.
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 beta-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.
Erster
Oran
O
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel. oerster@engineering.ucsb.edu
Liscovitch
Moti
M
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D008024:Ligands; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis
D017931:DNA Primers; D008024:Ligands; D004247:DNA