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Efficient detection, quantification and enrichment of subtle allelic alterations.

Chen J, Zhang X, Wang T, Li Z, Guan G, Hong Y - DNA Res. (2012)

Bottom Line: 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.Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.

ABSTRACT
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.

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Related in: MedlinePlus

Heteroduplex production and detection by TAGE and PAGE. (A) Flowchart showing heteroduplex production by PCR and heteroduplex detection by PAGE and TAGE. (B and C) Biallelic heteroduplex model assay. Plasmids containing a WT allele (WT1) and a mutant allele with an 18-bp deletion (D18) were mixed with indicated dilution factor for PCR. (B) TAGE detection. D18 with a dilution factor of 16 can be detected. (C) PAGE detection. D18 with a dilution factor of 256 can be detected. Size markers in base pairs are indicated to the left. Arrowheads denote a double band. Asterisks depict heteroduplexes. Hm, homoduplex; Ht, heteroduplex; PAGE, polyacrylamide gel electrophoresis; T7, T7 endonuclease I; TAGE, T7-digestion and agarose gel electrophoresis. This figure appears in colour in the online version of DNA Research.
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DSS023F1: Heteroduplex production and detection by TAGE and PAGE. (A) Flowchart showing heteroduplex production by PCR and heteroduplex detection by PAGE and TAGE. (B and C) Biallelic heteroduplex model assay. Plasmids containing a WT allele (WT1) and a mutant allele with an 18-bp deletion (D18) were mixed with indicated dilution factor for PCR. (B) TAGE detection. D18 with a dilution factor of 16 can be detected. (C) PAGE detection. D18 with a dilution factor of 256 can be detected. Size markers in base pairs are indicated to the left. Arrowheads denote a double band. Asterisks depict heteroduplexes. Hm, homoduplex; Ht, heteroduplex; PAGE, polyacrylamide gel electrophoresis; T7, T7 endonuclease I; TAGE, T7-digestion and agarose gel electrophoresis. This figure appears in colour in the online version of DNA Research.

Mentions: We are interested in versatile procedures for use in most ordinary laboratories without specialized equipment. Therefore, we developed a PAGE detection procedure, as is indicated in Fig. 1A, which can be streamlined as follows:Figure 1.


Efficient detection, quantification and enrichment of subtle allelic alterations.

Chen J, Zhang X, Wang T, Li Z, Guan G, Hong Y - DNA Res. (2012)

Heteroduplex production and detection by TAGE and PAGE. (A) Flowchart showing heteroduplex production by PCR and heteroduplex detection by PAGE and TAGE. (B and C) Biallelic heteroduplex model assay. Plasmids containing a WT allele (WT1) and a mutant allele with an 18-bp deletion (D18) were mixed with indicated dilution factor for PCR. (B) TAGE detection. D18 with a dilution factor of 16 can be detected. (C) PAGE detection. D18 with a dilution factor of 256 can be detected. Size markers in base pairs are indicated to the left. Arrowheads denote a double band. Asterisks depict heteroduplexes. Hm, homoduplex; Ht, heteroduplex; PAGE, polyacrylamide gel electrophoresis; T7, T7 endonuclease I; TAGE, T7-digestion and agarose gel electrophoresis. This figure appears in colour in the online version of DNA Research.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473374&req=5

DSS023F1: Heteroduplex production and detection by TAGE and PAGE. (A) Flowchart showing heteroduplex production by PCR and heteroduplex detection by PAGE and TAGE. (B and C) Biallelic heteroduplex model assay. Plasmids containing a WT allele (WT1) and a mutant allele with an 18-bp deletion (D18) were mixed with indicated dilution factor for PCR. (B) TAGE detection. D18 with a dilution factor of 16 can be detected. (C) PAGE detection. D18 with a dilution factor of 256 can be detected. Size markers in base pairs are indicated to the left. Arrowheads denote a double band. Asterisks depict heteroduplexes. Hm, homoduplex; Ht, heteroduplex; PAGE, polyacrylamide gel electrophoresis; T7, T7 endonuclease I; TAGE, T7-digestion and agarose gel electrophoresis. This figure appears in colour in the online version of DNA Research.
Mentions: We are interested in versatile procedures for use in most ordinary laboratories without specialized equipment. Therefore, we developed a PAGE detection procedure, as is indicated in Fig. 1A, which can be streamlined as follows:Figure 1.

Bottom Line: 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.Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus