在Dystrophin基因大内含子上定位缺失突变位点的策略  

Strategy of localizing the deletion mutation point in large introns of Dystrophin gene

作　　者：钟敏[1] 潘速跃[1] 蔡丽丹[1] 陆兵勋[1] 张国忠[1] 

机构地区：[1]南方医科大学南方医院神经内科,广东省广州市510515

出　　处：《中国临床康复》2006年第12期120-121,共2页Chinese Journal of Clinical Rehabilitation

基　　金：广东省自然科学基金(001032)~~

摘　　要：目的:探讨在Dystrophin基因大内含子上准确定位缺失突变位点的方法。方法:实验于2005-02/05南方医科大学南方医院神经内科实验室内完成。选择2例临床证实为男性假肥大型肌营养不良症患者,通过外显子聚合酶链反应检测证实其5’端断裂点分别位于Dystrophin基因庞大的44号和2号内含子。在44号和2号内含子上先各设计5对引物将内含子序列人为地分成长度大致均等的6个待查区域,经第一次聚合酶链反应检测确认断裂点所在区域后继续在该区域每3kb序列设计1对引物,在第2次聚合酶链反应步移法检测中若相邻2对内含子引物1对扩增出正常阳性结果而另1对扩增为阴性结果,即为内含子上断裂点的近似位置。结果:以5对初步定位引物进行聚合酶链反应检测后确定以上2例假肥大型肌营养不良症患者5’端断裂点位于44号内含子第5区和2号内含子第6区,在该2个区域进行第二次聚合酶链反应步移法检测,准确定位断裂点分别大致位于44号内含子178kb左右以及2号内含子153kb左右的位点处。结论:分次聚合酶链反应步移法定位Dystrophin基因大内含子上缺失突变位点的方法更为简便,对位于大内含子上基因缺失连接片段的准确快速克隆具有重要意义。AIM： To study the methods of accurately localizing the deletion mutation point in the large introns of dystrophin gene. METHODS： This experiment was carried out in Neurological Laboratory, Nanfang Hospital affiliated to Southern Medical University. Two Duchenne＇s muscular dystrophy （DMD） patients had been substantiated that their dystrophin gene 5＇ breakpoints were located in the large introns 44 and 2 separately by polymerase chain reaction （PCR） to the exons. Five primer pairs were designed in the 2 introns to divide every sequence into 6 approximately average regions. After the regions with breakpoints were detected by the first step PCR reactions we went on to design primer pairs every 3 kb sequence in the regions. Then the second step of PCR-based genome-walking method was carried out. If the PCR reactions with a primer pair were normal positive but the neighboring one was negative, the breakpoints in the introns were approximately localized. RESULTS： With the first step PCR reactions of 5 primer pairs in the 2 introns completed, that 5＇ breakpoints were located in the fifth region ofintron 44 and the sixth region of intron 2 was ascertained. After the second step of PCR-based genome.walking reactions in the 2 regions we accurately localized the breakpoints in the site of 178 kb around in intron 44 and 1 53 k b around in intron 2. CONCLUSION： Multiple-step PCR-based genome-walking method is simple and convenient to localize the deletion mutation point in the large introns of Dystrophin gene. It is of great importance using it to correctly and quickly clone the junction fragment that located in the large introns.

关 键 词：肌营养不良 杜氏/遗传学 内含子 基因缺失 

分 类 号：R394.1[医药卫生—医学遗传学]