Efficient Detection, Quantification and Enrichment of Subtle Allelic Alterations

Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing. Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations. Efficient detection of such minor alterations remains one of the challenges...

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Bibliographic Details
Published in:DNA research 2012-10, Vol.19 (5), p.423-433
Main Authors: Chen, Jianbin, Zhang, Xi, Wang, Tiansu, Li, Zhendong, Guan, Guijun, Hong, Yunhan
Format: Article
Language:English
Subjects:
Alleles
Animals
Biological and medical sciences
Endoribonucleases - metabolism
Fundamental and applied biological sciences. Psychology
Gene Targeting
Genes. Genome
Genetics of eukaryotes. Biological and molecular evolution
Heteroduplex Analysis - methods
Molecular and cellular biology
Molecular genetics
Oryzias
Transcription Factors - metabolism
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Summary:Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing. Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations. Efficient detection of such minor alterations remains one of the challenges in ZFN-mediated GT experiments. Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations. In a biallelic model, polyacrylamide gel electrophoresis (PAGE) is capable of detecting rare allelic variations in the form of DNA heteroduplexes at a high efficiency of ~0.4% compared with ~6.3% by the traditional T7 endonuclease I-digestion and agarose gel electrophoresis. In a multiple allelic model, PAGE could discriminate different alleles bearing addition or deletion of 1-18 bp as distinct bands that were easily quantifiable by densitometry. Furthermore, PAGE enables enrichment for rare alleles. We show for the first time that direct endogenous GT is possible in medaka by ZFN RNA injection, whereas PAGE allows for detection and cloning of ZFN-targeted alleles in adults arising from ZFN-injected medaka embryos. Therefore, PAGE is effective for detection, quantification and enrichment of multiple fine allelic differences and thus offers a versatile tool for screening targeted subtle gene alterations.
ISSN:1340-2838
1756-1663
DOI:10.1093/dnares/dss023