Qiuju Su1, Xiang Zhou1,2, Tianwen Wu3, Kui Li3, Wentao Xu4, Zhenyu Lin5, Ping Shen6, Bang Liu7,8. 1. Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China. 2. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China. 3. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. 4. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, 100083, Beijing, China. 5. MOE Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, 350002, Fujian, China. 6. Development Center for Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing, 100045, China. 7. Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China. liubang@mail.hzau.edu.cn. 8. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, China. liubang@mail.hzau.edu.cn.
Abstract
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.
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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