三种检测内源性基因修饰方法比较

doi:10.13560/j.cnki.biotech.bull.1985.2016.02.010

生物技术通报 ›› 2016, Vol. 32 ›› Issue (2): 76-83.doi: 10.13560/j.cnki.biotech.bull.1985.2016.02.010

三种检测内源性基因修饰方法比较

李炜杰^1，2, 杨娇², 何高明², 王立民¹, 皮文辉¹, 周平¹

1.新疆生产建设兵团绵羊繁育生物技术重点实验室，石河子 832000；2.石河子大学动物科技学院，石河子 832003

收稿日期:2015-05-15 出版日期:2016-02-24 发布日期:2016-02-25
作者简介:李炜杰，女，硕士，研究方向：动物临床疾病；E-mail：jie03945879198@163.com
基金资助:
国家自然科学基金项目(31360276)，兵团院士资金专项(2007JC19)，兵团国际合作项目(2013BC004)，新疆兵团绵羊繁育生物技术重点实验室项目(2013KLS01)，优质转基因肉羊新品种培育项目(2013ZX08008-003)

The Comparison of Three Methods of Monitoring Endogenous Gene Modification

LI Wei-jie^1，2, YANG Jiao², HE Gao-ming², WANG Li-min¹, PI Wen-hui¹, ZHOU Ping¹

(1.Key Laboratory of Sheep Breeding and Development Technology of Xinjiang Production and Construction Corps，Shihezi 832000；2.College of Animal Science and Technology，Shihezi University，Shihezi 832003)

Received:2015-05-15 Published:2016-02-24 Online:2016-02-25

摘要/Abstract

摘要： 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。

关键词: 基因组编辑, Surveyor核酸酶, T7 Endonuclease I, 高分辨率熔解曲线(HRM)

Abstract: In orderto 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 comparingthe 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.

Key words: genome editing, surveyor nuclease, T7 Endonuclease I, high resolution melting(HRM)

李炜杰, 杨娇, 何高明, 王立民, 皮文辉, 周平. 三种检测内源性基因修饰方法比较[J]. 生物技术通报, 2016, 32(2): 76-83.

LI Wei-jie, YANG Jiao, HE Gao-ming, WANG Li-min, PI Wen-hui, ZHOU Ping. The Comparison of Three Methods of Monitoring Endogenous Gene Modification[J]. Biotechnology Bulletin, 2016, 32(2): 76-83.

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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

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