Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor

文献类型： 外文期刊

第一作者： Su, Qiuju

作者： Su, Qiuju;Zhou, Xiang;Liu, Bang;Zhou, Xiang;Liu, Bang;Wu, Tianwen;Li, Kui;Xu, Wentao;Lin, Zhenyu;Shen, Ping

作者机构：

关键词： AS-PCR; LFNAB; Genome-editing; Genotyping; Pigs

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.316； 五年影响因子：2.357 ）

ISSN： 0301-4851

年卷期： 2021 年 48 卷 11 期

页码：

收录情况： SCI

摘要： Background Genome-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 Results The 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. Conclusion The 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.

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