Pyrosequencing 检测CYP2C9*3基因多态性方法的建立及其可靠性研究

中国临床药理学与治疗学 ›› 2009, Vol. 14 ›› Issue (7): 799-803.

Pyrosequencing 检测CYP2C9^*3基因多态性方法的建立及其可靠性研究

赵钢涛¹, 丁媛媛¹, 杨凡¹, 刘惠军², 姜楠¹, 许景峰¹

¹北京军区总医院药理科, 北京100700;
²黑龙江佳木斯市中心医院, 佳木斯154002, 黑龙江

收稿日期:2009-03-23 修回日期:2009-07-26 出版日期:2009-07-26 发布日期:2020-10-30
作者简介:赵钢涛, 女, 硕士, 主管药师, 主要从事药物基因组学相关研究。Tel:010-66721898 　E-mail:xiaotiaohan@163.com

Development of pyrosequencing method for the simultaneous and rapid detection of CYP2C9^*3 gene polymorphism and its reliability study

ZHAO Gang-tao¹, DING Yuan-yuan¹, YANG Fan¹, LIU Hui-jun², JIANG Nan¹, XU Jing-feng¹

¹Dept.of Pharmacology, General Hospital of Beijing Military Command, Beijing 100700, China;
²The Central Hospital of Jiamusi, Jiamusi 154002, Heilongjiang, China

Received:2009-03-23 Revised:2009-07-26 Online:2009-07-26 Published:2020-10-30

摘要/Abstract

摘要： 目的:建立基于焦磷酸测序技术(Pyrosequencing)的高通量的CYP2C9^*3 突变检测方法。方法:应用带有生物素标记的扩增引物, 经PCR扩增及Beads 分离, 制备该突变位点焦磷酸测序单链模板, 并在PyroMark ID 焦磷酸测序仪上进行焦磷酸测序, 以经典ABI 测序法测序结果作为标准对照, 观察批量分析的可靠性并批量检测220例人DNA 标本。结果:建立了基于焦磷酸测序技术的CYP2C9^*3 突变位点检测平台, 实现了DNA标本的高通量检测。能一次获得96 份DNA 的CYP2C9^*3 突变位点检测结果。经重复性检测和标准的ABI 测序可靠性检测, CYP2C9^*3 突变位点的突变检出率及重复率均达100%。结论:本方法可准确、高通量、快速检测CYP2C9^*3 突变,特别适宜该SNP 位点批量检测需要。

关键词: 焦磷酸测序, 多态性, 序列分析, CYP2C9

Abstract: AIM: To establish a pyrosequencingbased method for simultaneous and rapid detection of CYP2C9^*3 gene polymorphism.METHODS: With the results of standard ABI sequencing as a standard control, we observed and analyzed the reliability of the pyrosequencing method which involves the establishment of biotin-labeled primers, amplification of PCR, separation of Beads, preparation of pyrosequencing single-stranded template of mutational sites and sequencing of PyroMark ID in pyrosequencing apparatus by detected 220 cases of human DNA samples.RESULTS: We achieved the simultaneous and rapid detection platform of CYP2C9^*3 mutation of DNA samples based on Pyrosequencing technology, which can detect DNA samples with high-flux manner.The test results of the CYP2C9^*3 mutation of 96 DNA samples can be obtained each time with this detection platform.Compared with the standard ABI sequencing, the mutation detection rate and the repetition rate of CYP2C9^*3 mutation are 100% in this method.CONCLUSION: Pyrosequencing method is a accurate, high-throughput, rapid method for the CYP2C9^*3 mutation detection, especially suitable for the bulk detection of this mutation.

Key words: pyrosequencing, polymorphism, sequence analysis, CYP2C9

中图分类号:

R969

赵钢涛, 丁媛媛, 杨凡, 刘惠军, 姜楠, 许景峰. Pyrosequencing 检测CYP2C9^*3基因多态性方法的建立及其可靠性研究[J]. 中国临床药理学与治疗学, 2009, 14(7): 799-803.

ZHAO Gang-tao, DING Yuan-yuan, YANG Fan, LIU Hui-jun, JIANG Nan, XU Jing-feng. Development of pyrosequencing method for the simultaneous and rapid detection of CYP2C9^*3 gene polymorphism and its reliability study[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2009, 14(7): 799-803.

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