应用巢式PCR法检测猪单个胚胎中的基因突变

Establishment and Application of Nested PCR Method for Detecting Mutation in Single Cell

单胚胎基因突变检测技术是以单个胚胎的裂解产物为模板进行DNA或RNA分析的方法,该技术的建立将为在单细胞水平进行基因表达和突变检测提供简便而快捷的方法。本研究设计了2对巢式引物,以显微注射了Cas9mRNA和猪SS基因2个靶点的gRNA的四细胞期孤雌胚胎为试验材料,通过蛋白酶K对猪单个胚胎进行裂解后直接进行巢式PCR分型,并检测到猪胚胎中SS基因的删除突变。该方法可以最大限度减少假阴性和假阳性结果的出现,在猪基因突变检测中具有重要作用。

Gene mutation detection in single cell is a new method used for DNA or RNA analysis based on single cell lysis products,the establishment of this technology will provide an easy and quick way for gene expression and mutation detection in single cell level.In this paper,pig 4-cellstage parthenogenetic embryos microinjected with Cas9 mRNA and 2target gRNA from pig SS gene were used as experimental material,and two pairs of nested primers were designed for nested PCR.Using this detection method,long fragment deletion in SSgene was detected.This method can minimize the occurrence of false negative and false positive results,which may play an important role in the pig gene mutation detection.