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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, enrichment and quantification of multiple alleles. (A) PAGE and TAGE profiles of multiple alleles after successive rounds of PCR following gel recovery (dash frame). DNA mixture containing four alleles shown in lane 12 of Fig. 2B was used as template with mix ratios indicated in (C). (B) Relative intensity of different bands of each round. Round ‘0’ represent the initial percentages of each allele. (C) The number of clones and percentages of each allele from PCR products of each round. This figure appears in colour in the online version of DNA Research.
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DSS023F3: PAGE detection, enrichment and quantification of multiple alleles. (A) PAGE and TAGE profiles of multiple alleles after successive rounds of PCR following gel recovery (dash frame). DNA mixture containing four alleles shown in lane 12 of Fig. 2B was used as template with mix ratios indicated in (C). (B) Relative intensity of different bands of each round. Round ‘0’ represent the initial percentages of each allele. (C) The number of clones and percentages of each allele from PCR products of each round. This figure appears in colour in the online version of DNA Research.

Mentions: A final proof for targeted alterations in a ZFN-treated sample comes from sequence analysis. ZFN-mediated GT has been reported to occur in 1–3% cells of zebrafish embryos,34,35 where only one gene copy is targeted in most cases. Consequently, the targeted allele is usually present at ∼1% in total genomic DNA from a positive sample. Accordingly, the validation of GT is a time-consuming task in a ZFN-mediated GT experiment, because it requires preparing and sequencing several hundreds of recombinant colonies.34,35 The high resolution and band distinctness of intact PCR products on PAGE provoked us to develop a procedure of enriching for rare alleles, which is gel recovery and successive PCR (grsPCR). In grsPCR, Ht bands of first-round PCR are gel recovered, and DNA is released by diffusion from gel slices for use as template for successive rounds of PCR. To test this procedure, a DNA mixture of four alleles at defined ratio (column 1, Fig. 3C) was used for first round of PCR; Ht fractions were gel recovered collectively, and DNA released was used for second and third rounds of PCR (Fig. 3A), and PCR products were quantified by densitometry (Fig. 3B) and cloned for validation by sequencing (Fig. 3C). Upon two to three rounds of PCR, an increase in Ht fractions was indeed apparent on both PAGE and TAGE when compared with the Hm fractions (Fig. 3A). To quantify a change in intensity during grsPCR, various bands were defined and measured by densitometry (Supplementary Fig. S4A and B). According to the band patterns of this four-allelic model system as shown in Fig. 2B, major bands of the four alleles are identifiable, which permits intensity quantifications of the four alleles by combining two bands of each allele (Supplementary Fig. S4C). In this way, the intensity of dominant WT allele was found to be reduced considerably from 62.0% in the first PCR over 36.0–26.3%, while intensities of rare alleles increased after one and two rounds of grsPCR (Fig. 3B and Supplementary Fig. S4C). Cloning and sequencing led to a similar enrichment factor (Fig. 3C). After one single round of grsPCR, for example, the WT allele decreased from 79 to 55%, and rare alleles increased relatively. This verifies quantitative enrichment for rare alleles. Taken together, PAGE profile enables the quantification of each component, while grsPCR allows for the enrichment of multiple rare alleles.Figure 3.


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, enrichment and quantification of multiple alleles. (A) PAGE and TAGE profiles of multiple alleles after successive rounds of PCR following gel recovery (dash frame). DNA mixture containing four alleles shown in lane 12 of Fig. 2B was used as template with mix ratios indicated in (C). (B) Relative intensity of different bands of each round. Round ‘0’ represent the initial percentages of each allele. (C) The number of clones and percentages of each allele from PCR products of each round. 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

DSS023F3: PAGE detection, enrichment and quantification of multiple alleles. (A) PAGE and TAGE profiles of multiple alleles after successive rounds of PCR following gel recovery (dash frame). DNA mixture containing four alleles shown in lane 12 of Fig. 2B was used as template with mix ratios indicated in (C). (B) Relative intensity of different bands of each round. Round ‘0’ represent the initial percentages of each allele. (C) The number of clones and percentages of each allele from PCR products of each round. This figure appears in colour in the online version of DNA Research.
Mentions: A final proof for targeted alterations in a ZFN-treated sample comes from sequence analysis. ZFN-mediated GT has been reported to occur in 1–3% cells of zebrafish embryos,34,35 where only one gene copy is targeted in most cases. Consequently, the targeted allele is usually present at ∼1% in total genomic DNA from a positive sample. Accordingly, the validation of GT is a time-consuming task in a ZFN-mediated GT experiment, because it requires preparing and sequencing several hundreds of recombinant colonies.34,35 The high resolution and band distinctness of intact PCR products on PAGE provoked us to develop a procedure of enriching for rare alleles, which is gel recovery and successive PCR (grsPCR). In grsPCR, Ht bands of first-round PCR are gel recovered, and DNA is released by diffusion from gel slices for use as template for successive rounds of PCR. To test this procedure, a DNA mixture of four alleles at defined ratio (column 1, Fig. 3C) was used for first round of PCR; Ht fractions were gel recovered collectively, and DNA released was used for second and third rounds of PCR (Fig. 3A), and PCR products were quantified by densitometry (Fig. 3B) and cloned for validation by sequencing (Fig. 3C). Upon two to three rounds of PCR, an increase in Ht fractions was indeed apparent on both PAGE and TAGE when compared with the Hm fractions (Fig. 3A). To quantify a change in intensity during grsPCR, various bands were defined and measured by densitometry (Supplementary Fig. S4A and B). According to the band patterns of this four-allelic model system as shown in Fig. 2B, major bands of the four alleles are identifiable, which permits intensity quantifications of the four alleles by combining two bands of each allele (Supplementary Fig. S4C). In this way, the intensity of dominant WT allele was found to be reduced considerably from 62.0% in the first PCR over 36.0–26.3%, while intensities of rare alleles increased after one and two rounds of grsPCR (Fig. 3B and Supplementary Fig. S4C). Cloning and sequencing led to a similar enrichment factor (Fig. 3C). After one single round of grsPCR, for example, the WT allele decreased from 79 to 55%, and rare alleles increased relatively. This verifies quantitative enrichment for rare alleles. Taken together, PAGE profile enables the quantification of each component, while grsPCR allows for the enrichment of multiple rare alleles.Figure 3.

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