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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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PAGE detection of multiple alleles. (A) Partial sequences of five different alleles of the medaka gsdf gene. Additions are shown as a loop. Deletions are depicted by gaps. Bold letters indicate the cleavage site of ZFN. (B) PAGE profiles of different mixtures of the alleles. Mix ratios of the alleles are indicated above lanes. Dashed lines delineate the same heteroduplexes cross lanes. Asterisks highlight heteroduplexes. (C) Correlation between the number of alleles and total number of different PCR products which may show different bands on PAGE. Colour background depicts the positive correlation between the concentrations of each DNA strands and the corresponding band intensities. Capital letters, upper strands; small capital case letters, lower strands; underlining, homoduplexes; Hm, homoduplex; Ht, heteroduplex. This figure appears in colour in the online version of DNA Research.
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DSS023F2: PAGE detection of multiple alleles. (A) Partial sequences of five different alleles of the medaka gsdf gene. Additions are shown as a loop. Deletions are depicted by gaps. Bold letters indicate the cleavage site of ZFN. (B) PAGE profiles of different mixtures of the alleles. Mix ratios of the alleles are indicated above lanes. Dashed lines delineate the same heteroduplexes cross lanes. Asterisks highlight heteroduplexes. (C) Correlation between the number of alleles and total number of different PCR products which may show different bands on PAGE. Colour background depicts the positive correlation between the concentrations of each DNA strands and the corresponding band intensities. Capital letters, upper strands; small capital case letters, lower strands; underlining, homoduplexes; Hm, homoduplex; Ht, heteroduplex. This figure appears in colour in the online version of DNA Research.

Mentions: In the practice of ZFN-mediated GT experiments, additions and deletions may occur independently at different stages of development and in different cells, leading to the production of various different MT alleles. We furthered our experiments to establish and utilize a multi-allelic model system for the detection by PAGE. To this end, a set of five different alleles were produced within exon 1 of the medaka gsdf gene via ZFN-mediated GT in medaka embryos (see below). They were WT2, A18, D7, D6 and D1 (Fig. 2A). WT2 of 320 bp in length is the WT allele of gsdf (WT1 described above is the WT allele of the medaka nanog). These allelic sequences were cloned in pGEM-T Easy, and linearized plasmids were mixed at various ratios as described in Fig. 2B. As expected, PCR produced a single band on PAGE when DNA containing a single plasmid was used as template (lanes 1–5, Fig. 2B), while allelic variations, as tiny as 1-bp difference, were clearly distinguishable on PAGE as Hm and Ht forms (lanes 6, 7, 9, 11, Fig. 2B). Notably, PAGE is capable of simultaneous detection of multiple alleles as distinct bands (lanes 8, 10, 12, 13, Fig. 2B). To confirm that the Ht bands are not from artificial non-specific PCR products, PCR products manifesting single band in lanes 1–5 of Fig. 2B were mixed at the same ratio as indicated in Fig. 2B and then were subjected to a heteroduplex formation procedure. PAGE profiles (Supplementary Fig. S3) of these mixtures show exactly the same patterns as in Fig. 2B.Figure 2.


Efficient detection, quantification and enrichment of subtle allelic alterations.

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

PAGE detection of multiple alleles. (A) Partial sequences of five different alleles of the medaka gsdf gene. Additions are shown as a loop. Deletions are depicted by gaps. Bold letters indicate the cleavage site of ZFN. (B) PAGE profiles of different mixtures of the alleles. Mix ratios of the alleles are indicated above lanes. Dashed lines delineate the same heteroduplexes cross lanes. Asterisks highlight heteroduplexes. (C) Correlation between the number of alleles and total number of different PCR products which may show different bands on PAGE. Colour background depicts the positive correlation between the concentrations of each DNA strands and the corresponding band intensities. Capital letters, upper strands; small capital case letters, lower strands; underlining, homoduplexes; Hm, homoduplex; Ht, heteroduplex. 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

DSS023F2: PAGE detection of multiple alleles. (A) Partial sequences of five different alleles of the medaka gsdf gene. Additions are shown as a loop. Deletions are depicted by gaps. Bold letters indicate the cleavage site of ZFN. (B) PAGE profiles of different mixtures of the alleles. Mix ratios of the alleles are indicated above lanes. Dashed lines delineate the same heteroduplexes cross lanes. Asterisks highlight heteroduplexes. (C) Correlation between the number of alleles and total number of different PCR products which may show different bands on PAGE. Colour background depicts the positive correlation between the concentrations of each DNA strands and the corresponding band intensities. Capital letters, upper strands; small capital case letters, lower strands; underlining, homoduplexes; Hm, homoduplex; Ht, heteroduplex. This figure appears in colour in the online version of DNA Research.
Mentions: In the practice of ZFN-mediated GT experiments, additions and deletions may occur independently at different stages of development and in different cells, leading to the production of various different MT alleles. We furthered our experiments to establish and utilize a multi-allelic model system for the detection by PAGE. To this end, a set of five different alleles were produced within exon 1 of the medaka gsdf gene via ZFN-mediated GT in medaka embryos (see below). They were WT2, A18, D7, D6 and D1 (Fig. 2A). WT2 of 320 bp in length is the WT allele of gsdf (WT1 described above is the WT allele of the medaka nanog). These allelic sequences were cloned in pGEM-T Easy, and linearized plasmids were mixed at various ratios as described in Fig. 2B. As expected, PCR produced a single band on PAGE when DNA containing a single plasmid was used as template (lanes 1–5, Fig. 2B), while allelic variations, as tiny as 1-bp difference, were clearly distinguishable on PAGE as Hm and Ht forms (lanes 6, 7, 9, 11, Fig. 2B). Notably, PAGE is capable of simultaneous detection of multiple alleles as distinct bands (lanes 8, 10, 12, 13, Fig. 2B). To confirm that the Ht bands are not from artificial non-specific PCR products, PCR products manifesting single band in lanes 1–5 of Fig. 2B were mixed at the same ratio as indicated in Fig. 2B and then were subjected to a heteroduplex formation procedure. PAGE profiles (Supplementary Fig. S3) of these mixtures show exactly the same patterns as in Fig. 2B.Figure 2.

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