A highly efficient strategy to determine genotypes of genetically-engineered mice using genomic DNA purified from hair roots.
A highly efficient strategy to determine genotypes of genetically-engineered mice using genomic DNA purified from hair roots.
复制标题
使用从发根纯化的基因组 DNA 确定基因工程小鼠基因型的高效策略。
DOI：
10.1177/0023677216646088
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发表时间：
2017
影响因子：
2.4
通讯作者：
DiFulvio,Mauricio
中科院分区：
文献类型：
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作者：
Otaño-Rivera,Víctor;Boakye,Amma;Grobe,Nadja;Almutairi,MohammedM;Kursan,Shams;Mattis,LesanK;Castrop,Hayo;Gurley,SusanB;Elased,KhalidM;Boivin,GregoryP;DiFulvio,Mauricio
基因工程小鼠的基因分型是有效设计育种策略和鉴定突变小鼠的必要条件。这一过程依赖于对引入小鼠基因组序列的DNA标记的识别，这一任务通常使用聚合酶链反应（PCR）来完成。显然，基因分型的限制步骤是分离纯基因组DNA。分离小鼠DNA进行基因分型通常涉及痛苦的过程，如剪断尾巴、去除手指或打耳朵。虽然以前曾有人提出将头发采集作为基因组DNA的来源，但由于DNA产量低和担心污染，人们一直不愿意使用这种无痛苦的技术。在这项研究中，我们开发了一种简单，经济，高效的策略，使用Chelex®树脂从小鼠发根中纯化基因组DNA，适合进行基因分型。通过与使用市售试剂盒的标准DNA纯化方法进行比较，我们证明Chelex®有效且持续地从发根中纯化高质量DNA，最大限度地减少疼痛，缩短时间并降低与准确基因型测定相关的成本。因此，将发根与Chelex®结合使用是一种可靠且更人性化的DNA基因分型选择。
Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.