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《异柠檬酸脱氢酶1基因突变焦磷酸测序检测方法的建立-论文.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、2014正4月首都医科大学学报Apr.2014第35卷第2期JournalofCapitalMedicalUniversityV01.35No.2[doi:10.3969/j.issn.1006-7795.2014.02.016]·基础研究·异柠檬酸脱氢酶1基因突变焦磷酸测序检测方法的建立王丹慧蔡彦宁张燕莉高杰杨彩侠(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)【摘要】目的建立异柠檬酸脱氢酶1(isocitratedehydrogenase1,/DH1)基因突变的焦磷酸测序检测方法,确定该方
2、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/DH1突变的差异。方法构建携带野生型和突变型/DH1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/DH1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。【关键词】异柠檬酸脱氢酶1;焦磷酸测序法;
3、突变【中图分类号】Q754Establishmentofpyrosequencingmethodtodetectisocitratedehydrogenase1mutationsWangDanhui,CaiYanning,ZhangYanli,GaoJie,YangCaixia(DepartmentofNeurobiology,XuanwuHospital,CapitalMedicalUniversity,KeyLaboratoryforNeurodegenerativeDiseases,MinistryofEducation,BeijingGeria
4、tricMedicalResearchCenter,Be~iing100053,China)【Abstract】ObjectiveThepresentstudyaimedtoestablishapyrosequencingmethodfor/DH1mutationexamination,andquantifythesensitivityofthismethod.Furthermore,wetriedtocomparethediferencesbetweenthedirectsequencingandpyrosequencingforIDH1mutati
5、onexamination。MethodsPlasmidscarryingeitherwide—typeormutantIDH1genewereconstructed,andusedtooptimizethepyrosequeneingmethod.Theexactamountofmutantplasmidsmixedwithwide-typeplasmidswereservedastemplatesforthepyrosequeneingreactiontoquantifythesensitivityofpyrosequencingbasedmuta
6、tionexamination.Bothdirectsequencingandpyroquencingmethodswereusedtodetect/DH1mutationsin96gliomas.ResultsPyrosequencingdetectedaslowas2%/DH1mutationmixedinwide—typegene.Amongthe96gliomasamplesexamined.32.3%ofthesampleswereidentifiedascarryingIDH1mutationsbasedondirectsequencing
7、.while74.O%basedonpyrosequencingmethod.ConclusionPyrosequencingisareliableandsensitivemethodindetecting/DH1mutation,whichissuitableformoleculardiagnosis.【Keywords】isocitratedehydrogenase1;pyrosequencing;mutation胶质瘤是最常见的原发性颅内肿瘤,其中恶性胶质最早为Parsons等[2所发现,该研究显示约12%的多瘤的发病率为(5—8)/100万,5
8、年病死率在全身肿瘤中形性胶质母细胞瘤患者存在IDH1第132位精氨酸的仅次于胰腺癌和肺癌,位列
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