焦磷酸测序分析ABCG2基因多态性方法的建立

中国临床药理学与治疗学 ›› 2012, Vol. 17 ›› Issue (7): 779-784.

焦磷酸测序分析ABCG2基因多态性方法的建立

万子睿¹, 谢海棠², 俞竞¹, 刘英姿¹, 曾飞跃³, 王果¹

¹中南大学临床药理研究所,长沙 410078,湖南;
²皖南医学院弋矶山医院临床药学部,芜湖 241001,安徽;
³中南大学湘雅医院放射科,长沙 410008,湖南

收稿日期:2012-03-31 修回日期:2012-05-25 发布日期:2012-07-17
通讯作者: 王果,男,博士,副教授,主要研究方向为遗传药理和临床药理学。Tel: 0731-84805380-8407 E-mail:wangguo30@yahoo.com.cn
作者简介:万子睿,男,硕士研究生,主要研究方向为遗传药理学。Tel: 18684709759 E-mail: maxim_w@163.com
基金资助:
国家自然科学基金项目(81072706, 81173134);高等学校博士学科点专项科研基金资助课题(20090162120024);湖南省科技计划项目(2009JT3020);中央高校基本科研业务费(2012QNZT085,2012QNZT133)

Development of pyrosequencing method for detection of ABCG2 polymorphisms

WAN Zi-rui¹, XIE Hai-tang², YU Jing¹, LIU Ying-zi¹, ZENG Fei-yue³, WANG Guo¹

¹Institute of Clinical Pharmacology, Central South University, Changsha 410078, Hunan, China;
²Institute of Clinical Pharmacy and Pharmacology, Yijishan Hospital of Wanan Medical College, Wuhu 241001, Anhui, China;
³Department of Radiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China

Received:2012-03-31 Revised:2012-05-25 Published:2012-07-17

摘要/Abstract

摘要： 目的: 建立ABCG2 34G＞A和421C＞A单核苷酸多态性位点的焦磷酸测序方法。 方法: 抽取志愿者静脉血入EDTA抗凝管,常规苯酚/氯仿法制备全血gDNA,生物素标记引物对扩增多态位点目的片段,制备生物素标记单链模板,与测序引物退火结合后行焦磷酸测序。分析结果经毛细管电泳测序验证,并进行重复性检验。 结果: 本文建立了针对ABCG2 34G＞A和421C＞A多态性位点的焦磷酸测序方法,经毛细管电泳测序验证和重复性验证,结果准确可靠。ABCG2 34G和34A等位基因频率分别为 79.5%和 20.5%;421C和421A等位基因频率分别为 72.7%和 27.8%,均符合Hardy-Weinberg平衡。 结论: 本文建立的焦磷酸测序方法可准确、高通量、快速检测ABCG2 34G＞A和421C＞A单核苷酸多态性,并且特别适宜大样本量的临床及科研批量检测需要。

关键词: 药理学, 焦磷酸测序, ABCG2, 乳腺癌耐药蛋白, 单核苷酸多态性

Abstract: AIM: To establish a pyrosequencing based method for detection ABCG2 34G＞A and 421C＞A polymorphisms and to determine the frequency of these polymorphisms in healthy Chinese. METHODS: After preparation of gDNA from blood of 200 subjects, the target fragments were amplified by PCR, polymorphisms were detected on PyroMark ID by pyrosequencing technology. The reliability of pyrosequencing methods were validated by repeat tests and Sanger sequencing. RESULTS: We established a new pyrosequencing method to detect the ABCG2 34G＞A and 421C＞A polymorphisms polymorphisms in healthy Chinese. The detection rate and repetition rate were both 100%. The frequencies of ABCG2 34G and 34A alleles were 79.5% and 20.5%, respectively. The allele frequencies of ABCG2 421C and 421A were 72.7% and 27.8%, respectively. Genotype frequencies match the Hardy-Weinberg equilibrium. CONCLUSION: These pyrosequencing assays to detect ABCG2 polymorphisms are proved to be a rapid, accurate and high-throughput alternative to conventional methods, and it can be a preferred option in research and clinical application.

Key words: Pharmacology, Pyrosequencing, ABCG2, BCRP, SNPs

中图分类号:

R968

万子睿, 谢海棠, 俞竞, 刘英姿, 曾飞跃, 王果. 焦磷酸测序分析ABCG2基因多态性方法的建立[J]. 中国临床药理学与治疗学, 2012, 17(7): 779-784.

WAN Zi-rui, XIE Hai-tang, YU Jing, LIU Ying-zi, ZENG Fei-yue, WANG Guo. Development of pyrosequencing method for detection of ABCG2 polymorphisms[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2012, 17(7): 779-784.

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