焦磷酸测序技术检测k-ras基因突变方法的建立

中国临床药理学与治疗学 ›› 2015, Vol. 20 ›› Issue (4): 430-434.

焦磷酸测序技术检测k-ras基因突变方法的建立

周霞辉¹, 吴成就², 肖乐东², 孙立贤¹, 曹忠¹

¹长沙理工大学化学与生物工程学院,长沙 410004,湖南;
²中南大学湘雅医学检验所,长沙 410078,湖南

收稿日期:2014-01-28 修回日期:2014-06-23 发布日期:2015-05-07
通讯作者: 曹忠,男,教授,博士生导师,主要从事纳米生化分析与基因诊断识别的研究。Tel: 0731-84805380 E-mail: zhongcao2004@163.com
作者简介:周霞辉,女,硕士研究生,主要从事生化分析与药物基因组学方法学研究。Tel: 0731-85258736 E-mail: yiyezi169@163.com
基金资助:
国家自然科学基金(21275022, 21075011);湖南省科技计划(2013FJ3075);国家科技支撑计划(2012BAC17B01) 资助课题

Establishment of pyrosequencing method for detection of k-ras mutation

ZHOU Xia-hui¹, WU Cheng-jiu², XIAO Le-dong², SUN Li-xian¹, CAO Zhong¹

¹ School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410004, Hunan,China;
² Xiangya Medical Laboratory, Central South University, Changsha 410078, Hunan, China

Received:2014-01-28 Revised:2014-06-23 Published:2015-05-07

摘要/Abstract

摘要： 目的: 建立k-ras基因突变的焦磷酸测序方法,并分析该基因在结直肠癌中的突变率。方法: 制备10份卫生部临床检验中心2013年全国k-ras基因突变检测室间质评模板DNA,对焦磷酸测序技术检测k-ras基因突变方法进行验证;制备230例结直肠癌标本gDNA,应用PyroMark Q24焦磷酸测序仪进行k-ras基因的焦磷酸测序。结果: 建立了k-ras基因突变的焦磷酸测序新方法,检测正确率100%;230例结直肠癌标本中共检出基因突变型33例,总突变率 14.34% (33/230),其中12号密码子上34G>T的突变率为 1.74%(4/230),35G>A的突变率为 5.22% (12/230),35G>T的突变率为 3.04% (7/230),37G>A的突变率为 0.43% (1/230),13号密码子上38G>A的突变率为 3.91% (9/230)。结论: k-ras基因突变的焦磷酸测序新方法具有快速、准确和高通量的优点,适合于在科研和临床基因扩增检验中推广。

关键词: 结直肠癌, k-ras基因, 基因突变, 焦磷酸测序

Abstract: AIM: To establish a pyrosequencing method for detecting k-ras mutation and to investigate the frequency and characteristic of k-ras of patients with colorectal cancer.METHODS: A total of 10 DNA specimens from the ministry of health clinical test center of China for external quality assessment were prepared for k-ras mutation detecting method verification. A total of 230 DNA specimens from patients with colorectal cancer were used for k-ras mutation detection.RESULTS: The overall positive rate of k-ras gene mutations obtained by pyrosequencing was 14.34% (33/230), and the positive rate of mutations was 10% (23/230) at codon 12 [5.22% (12/230) for 35G>A, 3.04% (7/230) for 35G>T, 1.74% (4/230) for 34G>T] and 4.34% (10/230) at codon 13[0.43% (1/230) for 37G>A, 3.91% (9/230) for 38G>A].CONCLUSION: This pyrosequencing method to detect k-ras mutation is proved to be a rapid, accurate and high throughput method alternative to conventional method, and it can be a preferred option in research and clinical application.

Key words: colorectal cancer, k-ras gene, gene mutation, pyrosequencing

中图分类号:

R969

周霞辉, 吴成就, 肖乐东, 孙立贤, 曹忠. 焦磷酸测序技术检测k-ras基因突变方法的建立[J]. 中国临床药理学与治疗学, 2015, 20(4): 430-434.

ZHOU Xia-hui, WU Cheng-jiu, XIAO Le-dong, SUN Li-xian, CAO Zhong. Establishment of pyrosequencing method for detection of k-ras mutation[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2015, 20(4): 430-434.

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