Wang Kun, Mei Dong Yi, Liu Qian Nan, Qiao Xiao Huan, Ruan Wei Min, Huang Tian, Cao Geng Sheng
School of Life Science, Henan University, Kaifeng 475004, PR China.
School of Life Science, Henan University, Kaifeng 475004, PR China; Institute of Bioengineering, Henan University, Kaifeng 475004, PR China.
J Biotechnol. 2015 Nov 20;214:128-32. doi: 10.1016/j.jbiotec.2015.09.029. Epub 2015 Sep 28.
The indel-forming non-homologous end joining (NHEJ) pathway repairs double strand breaks in mammalian genomes, resulting in mutation formation following genome editing. Common techniques employed to identify these mutations include the amplified fragment length polymorphism (AFLP) and SURVEYOR assays, which are time consuming, laborious, and only offer a low level of sensitivity. An alternative to these approaches, which is examined in this study, is based on the quantitative PCR high-resolution melting (qPCR-HRM) curve analysis technique and offers simple implementation, is capable of handling large sample sizes, takes no more than 90 min, and produces sensitive results. Using the newly discovered RNA-guided CRISPR/Cas systems, the IL2RG and EMX1 genes were edited in the human 293T cell line in order to compare the mutation detection accuracies of the aforementioned methods. Genomic mutations were simulated by mixing mutated DNA fragments with normal fragments along a concentration gradient. The results of this comparative study showed that the HRM approach was both reproducible and accurate.
形成插入缺失的非同源末端连接（NHEJ）途径修复哺乳动物基因组中的双链断裂，导致基因组编辑后形成突变。用于鉴定这些突变的常用技术包括扩增片段长度多态性（AFLP）和SURVEYOR检测，这些技术耗时、费力，且灵敏度较低。本研究中考察的一种替代方法基于定量PCR高分辨率熔解（qPCR-HRM）曲线分析技术，具有操作简单、能够处理大量样本、耗时不超过90分钟且结果灵敏的特点。利用新发现的RNA引导的CRISPR/Cas系统，在人293T细胞系中对IL2RG和EMX1基因进行编辑，以比较上述方法的突变检测准确性。通过将突变的DNA片段与正常片段沿浓度梯度混合来模拟基因组突变。这项比较研究的结果表明，HRM方法具有可重复性和准确性。
