Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor.
Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor.
复制标题
通过等位基因特异性 PCR 和侧流核酸生物传感器对具有小基因组片段缺失的种系突变体进行快速视觉基因分型方法。
DOI：
10.1007/s11033-021-06734-x
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发表时间：
2021
影响因子：
2.8
通讯作者：
Liu Bang
中科院分区：
文献类型：
--
作者：
Su Qiuju;Zhou Xiang;Wu Tianwen;Li Kui;Xu Wentao;Lin Zhenyu;Shen Ping;Liu Bang
背景结合人工核酸酶的基因组编辑技术发展迅速，能够对大量生物的基因组DNA进行高效和精确的修饰。本研究旨在建立一种快速、灵敏、直观的小片段缺失突变种系基因分型方法。方法与结果以TALENS系统产生的MSTN基因缺失2个碱基和11个碱基的基因组编辑猪为材料，对所建立的等位基因特异性聚合酶链式反应(AS-PCR)和侧向流动核酸生物传感器(LFNAB)级联方法进行了验证。AS-PCR可以产生不同标签的产物，以区分基因组编辑的等位基因和野生型等位基因。应用LFNAB对AS-PCR产物进行目视检测，而无需使用额外的仪器。此外，AS-PCR和LFNAB级联方法能够准确、直观地区分MSTN基因小片段缺失的基因组编辑猪和检出限为0.1 ng的野生型猪。结论AS-PCR和LFNAB级联方法可以对基因组编辑小片段缺失突变的猪进行快速、直观的基因分型，为基因组编辑动物基因分型提供了一个平台。
BackgroundGenome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The present research aimed to establish a rapid, sensitive and visual method for genotyping of germline genome-edited mutants with small genomic fragment deletion.Methods and ResultsThe genome-edited pigs with 2-bp deletion and 11-bp deletion of Myostatin (MSTN) gene generated by TALENs system were used as test materials to check the proposed allele-specific PCR (AS-PCR) and lateral flow nucleic acid biosensor (LFNAB) cascade method. AS-PCR can produce products with different tags to distinguish genome-edited alleles and wild-type alleles. A LFNAB was applied to do visual detection of AS-PCR products without using additional instruments. Furthermore, we demonstrated that AS-PCR and LFNAB cascade could accurately and visually distinguish genome-edited pigs with small genomic fragment deletion of Myostatin (MSTN) gene and wild-type pigs with limit of detection (LOD) of 0.1 ng.ConclusionThe proposed AS-PCR and LFNAB cascade can do rapid and visual genotyping of genome-edited mutants with small genomic fragment deletion, serving as a platform for genome-edited animal genotyping.