一种基因编辑位点特异性PCR方法的开发和应用

Development and application of a gene editing site-specific PCR method

为了对基因组编辑产品进行精准定性和定量检测,以水稻SP1基因的编辑植株为材料,在编辑位点上下游设计通用引物,在编辑位点处设计基因编辑位点特异性TaqMan探针,建立了编辑位点特异性PCR方法。利用该方法可准确鉴定特异基因组编辑产品,检测灵敏度达到5~10拷贝,可在实时荧光PCR(qPCR)和微滴数字PCR(ddPCR)平台上对基因组编辑产品进行定量检测。由于数字PCR的微反应单元可消除野生型DNA对通用引物的竞争性消耗,与qPCR的定量结果相比,ddPCR定量结果具有更高的定量准确性。

Genome editing technology has been successfully used in agricultural breeding,it is urgent to establish accurate identification and quantification technology based on molecular characteristics of genome-edited products.In this study,SP1 gene-edited rice was used as materials to develop an editing site-specific PCR method by designing universal primer pair upstream and downstream of the editing sites,and gene editing site-specific TaqMan probes targeting at the editing sites.The results demonstrated that this method accurately identified specific genome-edited products with a detection sensitivity of 5-10 copies.The quantitative detection of genome-edited products was performed by both real-time PCR(qPCR)and droplet digital PCR(ddPCR).Since thousands of independent droplets of ddPCR might eliminate the competitive consumption of universal primers by wild-type DNA,the quantitative results of ddPCR showed higher quantitative accuracy than those of qPCR.