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Published on: September 20, 2016
Efficient detection, quantification and enrichment of subtle allelic alterations
Jianbin Chen1, Xi Zhang, Tiansu Wang
1Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
Summary
Polyacrylamide gel electrophoresis (PAGE) efficiently detects subtle gene alterations from zinc finger nuclease (ZFN) genome editing. This method allows for precise quantification and enrichment of rare alleles, advancing ZFN-mediated gene targeting research.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Gene targeting (GT) is crucial for genome editing, enabling precise DNA modifications.
- Zinc finger nucleases (ZFNs) facilitate minor allelic alterations, but their detection is challenging.
- Existing methods for detecting subtle genetic changes lack efficiency and sensitivity.
Purpose of the Study:
- To establish efficient procedures for detecting, quantifying, and enriching subtle gene alterations.
- To evaluate polyacrylamide gel electrophoresis (PAGE) as a tool for analyzing ZFN-mediated GT.
- To demonstrate the feasibility of endogenous gene targeting in medaka using ZFNs.
Main Methods:
- Polyacrylamide gel electrophoresis (PAGE) for DNA heteroduplex analysis.
- Comparison of PAGE with T7 endonuclease I-digestion and agarose gel electrophoresis.
- Application of PAGE in biallelic and multiple allelic models, including ZFN-injected medaka.
Main Results:
- PAGE detected rare allelic variations with high efficiency (~0.4%) in a biallelic model.
- PAGE distinguished and quantified alleles with 1-18 bp additions/deletions in a multiple allelic model.
- PAGE enabled enrichment of rare alleles and facilitated detection and cloning of ZFN-targeted alleles in medaka.
Conclusions:
- PAGE is a highly effective tool for the detection, quantification, and enrichment of subtle gene alterations.
- PAGE offers a versatile approach for screening targeted genetic modifications, overcoming limitations of traditional methods.
- This study demonstrates successful endogenous gene targeting in medaka using ZFNs, validated by PAGE analysis.
