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时间:2017-12-07
《基因突变敏感性分子开关检测β地中海贫血基因突变》由会员上传分享,免费在线阅读,更多相关内容在工程资料-天天文库。
1、178实用医学杂志2012年第28卷第2期基因突变敏感性分子开关检测l3地中海贫血基因突变兰芬郭紫芬邓坤龙廖端芳李凯摘要目的:采用高保真DNA聚合酶介导的基因突变敏感性分子开关准确、快速检测B地中海贫血。方法:首先提取正常人血液基因组DNA,根据已知p珠蛋白基因热点突变CD41—42、IVS2.654区域序列,分别设计突变序列扩增引物,利用低保真酶进行引物延伸反应,将其PCR产物克隆到pMD19一T载体,得到B地中海贫血常见的两个突变位点的基因突变克隆:CD41.42、IVs2—654,然后以这两个突变位点为检测靶点,分别设计3末端与野生型基因位点或突变基因位点配对的引物.硫化磷
2、酸修饰3末端并偶联高保真DNA聚合酶构成的基因突变敏感性分子开关.对含有相应突变的阳性质粒模板及正常人基因组DNA模板进行引物延伸反应,并通过凝胶成像系统对其进行分析。结果:当引物与模板完全配对时,引物被延伸,有PCR产物;当引物与模板不完全配对时.引物不被延伸.无PCR产物。结论:高保真DNA聚合酶介导的基因突变敏感性分子开关能准确、快速筛查0地中海贫血CD41—42、IVS2654突变,提示其在单基因遗传病的诊断中具有广阔的应用前景。关键词B地中海贫血;高保真聚合酶;硫化修饰引物DetectionofthegenemutationsofB-thalassemiabygenem
3、utation-sensitivemolecularswitchLANFen,GUOn,DENGKun—lun,LIA0Duan-fang,LIKai.InstituteofPharmacyandPharmacology,UniversityofSouthChina,Hengyang421001,ChinaCorrespondingauthor:LIKaiE—maiZ:kaili34@yahoo.com.ca【Abstract】0bjectiveTodetect3-thalassemiabytheapproachofgenemutation—sensitivemoleculars
4、witch.MethodsGenomicDNAwasextractedfromnormalblood.AccordingtothesequencesofknownhotspotmutationregionofCD41-42andIVS2-654in13一globingene,themutantsequenceprimerwasdesigned,andthenprimerextensionreactionwasperformedwithlow.fidelityenzyme.andthePCRproductswasclonedintopMD19.Tvector.Twokindsofc
5、lonesofB.thalassemiamutationgene(CD41.42andIVS2.654)wereobtained.Thenthesetwomutationregionwereusedasdetectiontarget,3·terminalprimerwhichwasmatchedwithwildtypeormutationgeneregionwasdesigned,andthe3'-terminalwasmodifiedwithsulfidephosphatetheandlinkedtogenemutation—sensitivemolecularswitchwh
6、ichwasconstructedbyhigh-fidelityDNApolymerase.ThepositiveplasmidtemplateincludingthetargetmutationandnormalgenomicDNAtemplatewereadministeredbyprimerextensionreaction,andwereanalyzedbygel—imaginesystem.ResultWhentheprimerwasfullymatchedwiththetemplate,theprimerwasextended;Whentheprimerwasnotf
7、ullymatchedwiththetemplate,theprimerwasnotextended.ConclusionGenemutation..sensitivemolecularswitchwhichwasconstructedbyhigh.fidelityDNApolymerasecouldaccuratelyandrapidlyscreenedthemutationofCD41—42andIVS2—654ofB—thalassemia,suggestingthatit
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