A New Method for Identifying and Genotyping MSTN Gene-edited Cattle

doi:10.13560/j.cnki.biotech.bull.1985.2018-0748

Abstract

Abstract: Gene editing is a critical technique to generate genetically modified animals;however,it is hard to identify and genotype gene-edited animal because deleted fragment during editing is quite small. In this study,functional nucleic acid PCR（FNA-PCR）technique was established for identifying and genotyping MSTNgene-edited cattle. Based on 2 alleles for edited sites,2 different forward primers and one common reverse primer were designed. One forward primer was used to identify the allele of wild type samples,its 3'end was terminated at the edited site,and functional nucleic acid joint was designed to its 5'end. The other forward primer was used to detect MSTN gene-edited cattle in which the 3'end extended several nucleotides beyond the gene-edited site. This unique design allowed the amplified allele sizes of gene-edited and wild-type varied. By optimizing PCR reaction system and conditions,a novel FNA-PCR method,3 different genes were accurately genotyped in one PCR reaction,was established. FNA-PCR was high in sensitivity with detection limit 0.1 ng of MSTN gene-edited cattle genome DNA. In conclusion,FNA-PCR is a simple,accurate and rapid for identifying gene-edited animal and genotyping those small-fragment-deletion genes.

Key words: gene-edited cattle, FNA-PCR, genotyping, MSTN

SU Qiu-ju, ZHOU Xiang, LI Guang-peng, BAI Chun-ling, XU Wen-tao, LIU Bang. A New Method for Identifying and Genotyping MSTN Gene-edited Cattle[J]. Biotechnology Bulletin, 2019, 35(4): 208-212.

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