MELAS和MERRF综合征相关mtDNA突变位点检测集成芯片的建立  

Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF syndromes

作　　者：陈刚[1,2] 李伟[1] 杜卫东[1] 曹慧敏[2] 汤华阳[1] 唐先发[1] 孙中武[3] 赵辉[2] 金庆辉[2] 赵建龙[2] 张学军[1] 

机构地区：[1]安徽医科大学省部共建教育部重要遗传病基因资源利用重点实验室,安徽省基因研究重点实验室,合肥230032 [2]中国科学院上海微系统与信息技术研究所,上海200050 [3]安徽医科大学第一附属医院神经内科,合肥230022

出　　处：《遗传》2008年第10期1279-1286,共8页Hereditas（Beijing)

基　　金：国家自然科学基金项目(编号:30470591);安徽省自然科学基金项目(编号:050430716);安徽省“十五”高校学科拔尖人才基金项目资助~~

摘　　要：文中建立了一种新型的寡核苷酸芯片,用于线粒体脑肌病伴高乳酸血症和卒中样发作(Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes,MELAS)和肌阵挛性癫痫伴发不规整红纤维(Myoclonic epilepsy with ragged red fibers,MERRF)线粒体DNA所有已知突变位点的集成检测。将31对allele位点特异性的寡核苷酸探针包被在醛基修饰的载玻片表面,以多重不对称PCR方法制备Cy5荧光标记靶基因。利用此芯片对5例MELAS患者、5例MERRF患者及20例健康对照进行筛查,结果发现,MELAS患者均为MT-T1基因A3243G突变;在MERRF患者组,MT-TK基因A8344G突变4例,T8356C突变1例;健康对照组均未发现31种相关mtDNA突变。芯片检测与DNA测序结果完全一致。结果表明,这种寡核苷酸芯片可以对MELAS和MERRF综合征已知突变位点进行同步快速检测,具有较高的灵敏度和特异性。这一模式的基因芯片经过适当改装后也可用于其他人类线粒体疾病的基因诊断。We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitoehondrial DNA mutations associated with MELAS （Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes） and MERRF （Myoclonic epilepsy with ragged red fibers）. Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-1abled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR （MAP） method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip com- bined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

关 键 词：寡核苷酸芯片 线粒体DNA 突变 MELAS综合征 MERRF综合征 

分 类 号：R746[医药卫生—神经病学与精神病学]