摘要
目的探讨高保真聚合酶介导的分子开关突变检测技术对于筛查宫颈癌患者表皮生长因子受体(epidermal growth factor receptor,EGFR)基因G719S、T790M突变的价值,并分析上述突变与宫颈癌的相关性,以指导小分子酪氨酸激酶抑制剂类药物(tyrosine kinase inhibitor,TKI)的合理使用。方法设计两个位点的野生型与突变型序列的特异性引物,并硫化修饰引物的3′末端。以实验室构建的重组质粒为对照模板,进行高保真酶介导的PCR反应。将上述方法应用于临床样本的突变检测,并辅以测序验证。结果在80例宫颈癌组织以及31例癌旁组织的DNA样本中均未检测出EGFR基因的G719S、T790M突变,与DNA测序的结果一致。病例组的基因型分布频率与对照组的差异无统计学意义(P>0.05)。结论建立了检测EGFR基因G719S、T790M突变的分子开关技术平台。EGFR基因的G719S、T790M突变可能与宫颈癌无关。
Objective To establish a rapid and accurate "on/off" switch technique consisted of 3′ phosphorothioate-modified allele-specific primers and exo+ polymerase to screen the G719S and T790M mutations of epidermal growth factor receptor (EGFR) gene. The switch was used to identify cervical cancer patients who are sensitive to tyrosine kinase inhibitor (TKI). Methods Allele-specific primers targeting recombinant wild-type and mutation-type templates were designed with 3′ terminal phosphorothioate modification. Two-directional primer extension was carried out using Pfu polymerase. The G719S and T790M mutations were detected by the technique among cervical cancer tissues. The results were verified by Sanger sequencing. Results No mutation was detected among the 80 cervical cancer cases, and the results were consistent with that of Sanger sequencing. No significant difference was found between the frequencies of the G719S and T790M mutations between the patient and the control groups (P>0.05). Conclusion A sensitive on/off switch technique for detecting the two EGFR mutations was established. The G719S and T790M mutations are not associated with cervical cancer.
作者
向花花
周晶
彭华
董巍檑
龚咏晴
张宏全
郭紫芬
Xiang Huahua;Zhou Jing;Peng Hua;Dong Weilei;Gong Yongqing;Zhang Hongquan;Guo Zifen(Institute of Pharmacy and Pharmacology, Hunan Provincial Cooperative Innovation Center for Molecular Target New Drug Research, University of South China, Hengyang, Hunan 421001, China;Department of Obstetrics and Gynecology, the First Affiliated Hospital of University of South China, Hengyang, Hunan 421001, China)
出处
《中华医学遗传学杂志》
CAS
CSCD
2019年第4期376-379,共4页
Chinese Journal of Medical Genetics
基金
湖南省卫计委科研计划课题(C2016083)
湖南省自然科学基金(2018JJ2350).
