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Nucleic Acids Res. 2015 May 19;43(9):e59. doi: 10.1093/nar/gkv126. Epub 2015 Mar 9.

Fast and sensitive detection of indels induced by precise gene targeting.

Author information

1
Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark.
2
Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark.
3
Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark.
4
Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark.
5
Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark epb@sund.ku.dk.

Abstract

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

PMID:
25753669
PMCID:
PMC4482057
DOI:
10.1093/nar/gkv126
[Indexed for MEDLINE]
Free PMC Article

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