三种检测内源性基因修饰方法比较

The Comparison of Three Methods of Monitoring Endogenous Gene Modification

为获得准确的突变信息,除直接测序外,实验初步确定了3种人工核酸酶生物学活性检测方法。利用Surveyor nuclease、T7E1(T7 Endonuclease 1)和HRM(High resolution melt),均以变性退火突变型和野生型DNA序列形成扭曲的双螺旋DNA(distorted duplex DNA)为基础的3种人工核酸酶生物学活性检测方法,确定目标位点是否发生突变。实验成功检测出作用于绵羊MNST基因第一外显子的CRISPR/Cas9和第三外显子的TALEN目标位点发生突变,并对3种检测方法的结果和特点进行了分析比较,得出3种检测方法的优缺点,为实验室分析确定细胞利用非同源末端连接修复DNA双链断裂结果提供参考。

In order to obtain accurate mutation information, 3 methods of monitoring the biological activities of artificial nuclease are preliminary determined excluding direct sequencing. The 3 methods of Surveyor nuclease, T7E1（T7 Endonuclease 1）and HRM（High resolution melting）are all based on the principle of forming the twisted duplex DNA from denaturized annealing mutant and wild type DNA sequence, and whether or not target sites were mutated were determined. The results showed that using the 3 detection methods, the mutations at the target sites of exon 1＇s CRISPR/Cas9 and exon 3＇s TALEN in ovine gene MNST were successfully detected. Analyzing and comparing the results and characteristics of the 3 methods, the advantages and disadvantages of them were obtained, which provided a reference for the analysis and identification of results while the cells uses non-homologous ends to join and repair DNA double strand breaks. In conclusion, the results by Surveyor, T7E1 and HRM show that CRISPR/Cas9 and TALEN＇s target sites are mutated.