高分辨率熔解曲线在CRISPR基因编辑检测和基因型鉴定中的应用  

Application of high resolution melting curve in CRISPR gene editing and genotyping

作　　者：贺文 吴婉婉 陆平[1] 薛志刚[1] 盛哲津[2] 祝献民[2] HE Wen;WU Wan-wan;LU Ping;XUE Zhi-gang;SHENG Zhe-jin;ZHU Xian-min(Tongji University School of Medicine,Shanghai 200092,China;College of Life Science and Technology,Tongji University,Shanghai 200092,China)

机构地区：[1]同济大学医学院,上海200092 [2]同济大学生命科学与技术学院,上海200092

出　　处：《同济大学学报（医学版）》2018年第4期17-21,共5页Journal of Tongji University(Medical Science)

基　　金：国家自然科学基金重点项目(81430026);上海市科委基础研究领域项目(16JC1404700)

摘　　要：目的建立一种基于PCR和在线高分辨率熔解曲线(high-resolution melting curve,HRM)分析工具的检测方法,实现对DNA序列微小差异的快速检测。方法在CRISPR基因编辑检测和基因型鉴定中,采用两步PCR,分别对目的片段进行扩增和富集,并通过免费在线软件对HRM进行分析。结果 HRM分析结果显示,本实验所设计的CRISPR慢病毒系统未能对COS7细胞中的RPE65基因进行有效的基因编辑;而在瘦素突变大鼠的子代基因型中,HRM可以明显区分杂合子和纯合子。结论 HRM能够快速鉴定CRISPR编辑和区分基因型。Objective To establish a new method based on online high.resolution melting （HRM）analysis and PCR to promptly verify the DNA mismatches. Methods In detection of CRISPR genomeediting and genotyping, two.step PCR was used to amplify and enrich the target fragments. HRM wasanalyzed by free online software, then the mismatches caused by CRISPR were detected and thegenotypes were identified. Results HRM analysis showed that the CRISPR lentivirus system did notefficiently modified RPE65 gene in COS7 cells. In genotyping the progeny of leptin.mutant rats,HRM apparently distinguished heterozygotes from homozygotes. Conclusion HRM allows rapididentification of CRISPR editing and genotyping.

关 键 词：高分辨率熔解曲线 CRISPR 聚合酶链式反应 基因型 

分 类 号：Q331[生物学—遗传学]