A Rapid and General Assay for Monitoring Endogenous Gene Modification

• DOI: 10.1007/978-1-60761-753-2_15
• Publication Date: 2010-08-02T20:14:58Z
• Authors: Dmitry Y. Guschin; Adam J. Waite; George E. Katibah; Jeffrey C. Miller; Michael C. Holmes; Edward J. Rebar
• Keywords: 0301 basic medicine; 0303 health sciences; 03 medical and health sciences; DNA Repair; Animals; Humans; Biological Assay; DNA Breaks, Double-Stranded; Electrophoresis, Polyacrylamide Gel; Zinc Fingers; Endonucleases; Models, Biological; Polymerase Chain Reaction

The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.