异柠檬酸脱氢酶1基因突变焦磷酸测序检测方法的建立  

Establishment of pyrosequencing method to detect isocitrate dehydrogenase 1 mutations

作　　者：王丹慧[1] 蔡彦宁[1] 张燕莉[1] 高杰[1] 杨彩侠[1] 

机构地区：[1]首都医科大学宣武医院神经生物学研究室教育部神经变性病重点实验室北京市老年病医疗研究中心,北京100053

出　　处：《首都医科大学学报》2014年第2期219-224,共6页Journal of Capital Medical University

基　　金：国家自然科学基金(81071011);教育部新世纪优秀人才计划(NCET-10-013);北京市卫生系统高层次人才基金(008-0023)~~

摘　　要：目的建立异柠檬酸脱氢酶1(isocitrate dehydrogenase 1,IDH1)基因突变的焦磷酸测序检测方法,确定该方法的检测灵敏度。分析焦磷酸测序法与直接测序法对于鉴定IDH1突变的差异。方法构建携带野生型和突变型IDH1基因的质粒,使用质粒优化焦磷酸测序方法。使用已知比例的野生型和突变型质粒作为模版,确定突变的检测灵敏度。针对96例胶质瘤患者手术切除标本的基因组DNA,分别使用直接测序法和焦磷酸测序法,鉴定IDH1基因突变类型,并比较。结果使用焦磷酸测序能够检测到低至2%的IDH1突变。直接测序检出突变阳性率为32.3%、焦磷酸测序检出突变阳性率为74.0%。结论焦磷酸测序法检测IDH1基因突变灵敏可靠,适合临床分子诊断。Objective The present study aimed to establish a pyrosequencing method for IDH1 mutation examination, and quantify the sensitivity of this method. Furthermore, we tried to compare the differences between the direct sequencing and pyrosequencing for IDH1 mutation examination. Methods Plasmids carrying either wide-type or mutant IDH1 gene were constructed, and used to optimize the pyrosequencing method. The exact amount of mutant plasmids mixed with wide-type plasmids were served as templates for the pyrosequencing reaction to quantify the sensitivity of pyrosequencing based mutation examination. Both direct sequencing and pyroquencing methods were used to detect IDH1 mutations in 96 gliomas. Results Pyrosequencing detected as low as 2 % IDH1 mutation mixed in wide-type gene. Among the 96 glioma samples examined, 32. 3% of the samples were identified as carrying IDH1 mutations based on direct sequencing, while 74. 0% based on pyrosequencing method. Conclusion Pyrosequencing is a reliable and sensitive method in detecting IDH1 mutation, which is suitable for molecular diagnosis.

关 键 词：异柠檬酸脱氢酶1 焦磷酸测序法 突变 

分 类 号：Q754[生物学—分子生物学]