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J Genet Genomics. 2016 May 20;43(5):341-8. doi: 10.1016/j.jgg.2016.04.005. Epub 2016 May 4.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis.

Author information

1
Institut National de la Santé Et de la Recherche Médicale UMR 1064-Institut Transplantation Urologie-Néphrologie, Centre Hospitalier Universitaire de Nantes, Nantes 44093, France; Transgenesis Rat ImmunoPhenomic Platform, Structure Fédérative de Recherche François Bonamy, Centre National de Recherche Scientifique UMS3556, Nantes 44093, France.
2
Institut National de la Santé Et de la Recherche Médicale UMR 1064-Institut Transplantation Urologie-Néphrologie, Centre Hospitalier Universitaire de Nantes, Nantes 44093, France; GenoCellEdit, INSERM UMR 1064-ITUN, CHU Nantes, Nantes 44093, France.
3
Institut National de la Santé Et de la Recherche Médicale UMR 1064-Institut Transplantation Urologie-Néphrologie, Centre Hospitalier Universitaire de Nantes, Nantes 44093, France; Transgenesis Rat ImmunoPhenomic Platform, Structure Fédérative de Recherche François Bonamy, Centre National de Recherche Scientifique UMS3556, Nantes 44093, France. Electronic address: laurent.tesson@univ-nantes.fr.

Abstract

The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

KEYWORDS:

Capillary electrophoresis; Genome editing; Genotyping; Heteroduplex mobility assay

PMID:
27209567
DOI:
10.1016/j.jgg.2016.04.005
[Indexed for MEDLINE]

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