Efficient detection, quantification and enrichment of subtle allelic alterations.
Efficient detection, quantification and enrichment of subtle allelic alterations.
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DOI：
10.1093/dnares/dss023
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发表时间：
2012-10
期刊：
影响因子：
--
通讯作者：
Hong Y
中科院分区：
文献类型：
--
作者：
Chen J;Zhang X;Wang T;Li Z;Guan G;Hong Y
基因打靶（GT）可以将细微的改变引入特定的基因座，是基因组编辑的有力工具。工程化的锌指核酸酶（ZFN）对于产生微小等位基因改变是有效的。这种微小改变的有效检测仍然是ZFN介导的GT实验中的挑战之一。在这里，我们报告的程序的建立，允许有效的检测，定量和富集这种微妙的变化。在双等位基因模型中，聚丙烯酰胺凝胶电泳（PAGE）能够以高达10.4%的效率检测DNA异源双链形式的罕见等位基因变异，而传统的T7内切酶I消化和琼脂糖凝胶电泳的检测效率为10.3%。在多等位基因模型中，PAGE可以将带有1 - 18 bp的添加或缺失的不同等位基因区分为容易通过光密度法定量的不同条带。此外，PAGE能够富集稀有等位基因。我们首次表明，直接内源性GT是可能的青鳉通过ZFN RNA注射，而聚丙烯酰胺凝胶电泳允许检测和克隆ZFN注射青鳉胚胎中产生的成人ZFN靶向等位基因。因此，PAGE对于检测、定量和富集多个精细等位基因差异是有效的，并且因此提供了用于筛选靶向的细微基因改变的通用工具。
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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影响因子：
4.8
作者：
Li, GM;Wang, HX;Romano, LJ
通讯作者：
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.1117984109
发表时间：
2012-05-22
影响因子：
11.1
作者：
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影响因子：
2.6
作者：
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通讯作者：
Schartl, M