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A Rapid and General Assay for Monitoring Endogenous Gene Modification

  • Dmitry Y. Guschin
  • Adam J. Waite
  • George E. Katibah
  • Jeffrey C. Miller
  • Michael C. Holmes
  • Edward J. Rebar
Part of the Methods in Molecular Biology book series (MIMB, volume 649)

Abstract

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.

Key words

Zinc finger nuclease (ZFN) designed zinc finger proteins non-homologous end joining (NHEJ) Surveyor nuclease Cel 1 genome modification 

Notes

Acknowledgments

We thank Elo Leung, Xiangdong Meng, Sarah Hinkley, and Lei Zhang for help with the design and assembly of ZFNs; Jianbin Wang and Geoff Friedman for transfections; and Philip Gregory, Susan Abrahamson, and Lei Zhang for helpful comments on the manuscript.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Dmitry Y. Guschin
    • 1
  • Adam J. Waite
    • 1
    • 2
  • George E. Katibah
    • 1
  • Jeffrey C. Miller
    • 1
  • Michael C. Holmes
    • 1
  • Edward J. Rebar
    • 1
  1. 1.Sangamo BioSciences, Inc.RichmondUSA
  2. 2.Fred Hutchinson Cancer Research CenterSeattleUSA

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