β地中海贫血与先天性软骨发育不全诊断性基因芯片的构建.pdf

第三军医大学硕士学位论文 5 -地中海贫血与先天性软骨发育不全诊断性 基因芯片的构建 摘 要 -地中海贫血 beta-thalassemia)是一组由于珠蛋白肽链合成减少或缺乏而引起的以贫血为特征的遗传性血液病,发病率为0.65%我国南方的高发区约有2-3%的人是突变基因的携带者目前发现的突变类型达到170余种其中在中国发现的有21种-地中海贫血常见的突变包括CD41-42 (41.6%)CD17(18%)TATA-28 (8.0%)CD71-72(+A) (3.9%)TATA-29 (1.2%)集中在一段长约600bp的碱基链上先天性软骨发育不全achondroplasiaACH)是由于软骨内成骨缺陷所致的一种最常见的常染色体显性遗传性侏儒新生儿发病率为1-15/10万1994年发现ACH的致病基因定位于4p16.3 随后的研究发现ACH是由于成纤维细胞生长因子受体3 fibroblast growth factor 3FGFR3)跨膜区基因突变所致目前报道的突变97%以上来自FGFR3基因第10外显子的1138位核苷酸其中95%为G-A的碱基置换其余为G-C的转换通过产前基因诊断包括胚胎植入前诊断Preimplantation Genetic DiagnosisPGD)判断胎儿是否携带致病基因从而防止带病胎儿出生是预防上述两种疾病的有效办法国内外有条件的实验室已经开展了对单种疾病的胚胎植入前诊断但受诊断方法和标本量的限制尚未见能同时诊断多种疾病的检测技术报道基因芯片技术是近年发展起来的一种新的基因检测技术该技术的出现为同时诊断两种以上的遗传病提供了可能在已经建立了单细胞水平基因诊断技术的基础上本研究针对上述两种疾病的常见基因突变位点采用寡核苷酸探针和生物素标记显色技术建立一种能快速准确地同时检测和鉴定两种疾病特定类型的基因突变位点操作简便适合临床使用的基因芯片检测技术 方法与结果 1 通过查阅文献并在 GENEBANK 上检索获得-地中海贫血和 ACH 基因突变所在的 DNA 片段序列设计相应的引物, 进行了两种疾病特异性 DNA 片段的扩增产物 1 包含-地中海贫血 CD41-42 CD17 TATA-28 等突变位点 占突变频率的 72.7%600bp)产物 2包含 ACH 主要突变位点占突变频率的 99%1264bp)并对扩增条件进行优化使两个扩增反应能在相同的条件下完成 2设计 4 对针对各个突变位点特异性寡核苷酸探针对探针的特异性可靠性第三军医大学硕士学位论文 6 进行检测确定了杂交条件结果显示探针间 Tm 值相差小于 2各探针在 G+C 含量发夹结构自身二聚体以及重复碱基数等指标均符合寡核苷酸探针设计要求所选用探针特异性强具有相同的杂交条件能够区分仅相差一个碱基的靶序列 3将筛选出的检测探针阳性对照探针以及阴性探针点在尼龙膜上验证后制成玻璃芯片用于样品的检测相应检测探针点及阳性对照均有杂交结果阴性对照和其他探针点均无显色结果背景清晰 4为确定最佳点样探针浓度进行了探针浓度对探针固定效率和杂交效率的影响研究结果表明点样探针浓度为 1000-2000nmol/L 时可获得较好的固定率和杂交效率因此将制备芯片的探针浓度确定为 1500nmol/L 5为确定最适杂交温度分别采用 40455055为杂交温度发现在 45以下杂交时探针特异性不强50以上则灵敏度明显下降 在 48时实验结果最为稳定重复 10 次实验未出现假阳性及假阴性结果 6芯片杂交后的检测结果可以用肉眼观察选用 TMB 与 DAB 显色在尼龙膜上杂交时TMB 与 DAB 均能获得较好的显色结果但 TMB 在光照和有氧条件下易分解在玻璃上杂交时DAB 不论是显色灵敏度还是保存时间均明显优于 TMB 7用该芯片对 3 例-地中海贫血和 3 例临床诊断考虑为软骨发育不全的病例和6 例正常样本成功进行了基因诊断与其他基因诊断方法结果一致 结论 1采集-地中海贫血与先天性软骨发育不全患儿的血液标本构建了能够同时检测-地中海贫血与先天性软骨发育不全的常见突变位点的基因芯片 建立了同时诊断两种遗传病的基因诊断技术为产前诊断先天遗传性疾病控制出生缺陷提供了技术支持经 MEDLINE 检索国内外均未见报道 2该芯片制作成本低重复性好特异性高能满足临床标本检测的要求芯片检测时不需要特殊设备操作方法简单在三级以上医院均可开展具有良好的临床应用前景 关键词DNA 芯片 基因芯片 -地中海贫血 先天性软骨发育不全 生物素 第三军医大学硕士学位论文 2DNA chip construction for diagnosing ββ-thalathemia and achondraplasia Abstract β-thalassaemia is one of the autosomal genetic blood diseases characterized by absent or decreased production of normal beta hemoglobin. The incidence of β-thalassaemia is 0.65%, and the carriers account for 2%-3% in high incidence areas in South China. Over 170 types of mutations have been found in the whole world, among which, 21 are found in China. The most common mutations including CD41-42 (41.6%), CD17(18%), TATA-28 (8.0%), CD71-72(+A)(3.9%), TATA-29(1.2%) are at the same segment which is only 600 bps. Achondroplasia (ACH) is a common autosomal dominant inheritant dwarf caused by defection of cartilaginous ossification. The incidence of the neonate is 1-15/106. The pathogenic gene of ACH was located on 4p16.3 in 1994. Following researches revealed that it was caused by the mutation of the transmembrane dominant of fibroblast growth factor 3 (FGFR3) gene. 97% of the reported mutations are substitution of the 1138th base in the tenth exon. 95% is G-A and the others is G-C. Prenatal genetic diagnosis including preimplantation genetic diagnosis (PGD) has been proven to be an effective strategy for preventing the incidence of those diseases. Successful PGD on each single disease had been reported. But there was not any successful test which diagnosis more than two diseases at the same time for the restriction of detection technology and the sample content. Gene chip is a new technology which was developed by the recent years. This technology made it possible to diagnosis more than one genetic diseases with only one chip. In order to diagnosis β-thalassaemia and ACH at the same time, we successfully built a gene chip for gene mutation detection, which is simple, fast, accurate and reliable to the clinlic application after research on genetic diagnosis by single cell. The oligonucleotide probes with regard to the special mutations of these two diseases were designed by certain software and biotin labeling technology were used in our research. Methods and Results : 1. According to the literature and the GENEBANK database, the mutant gene 第三军医大学硕士学位论文 3sequence were acquired and the primers were designed to amplify certain DNA segaments. Product 1 contained CD41-42, CD17 and TATA-28 of β-thalassaemia(accounting for 72.7% of the mutation frequency, 600bp); Product 2 contained the main mutation of ACH(accounting for 99% of the mutation frequency, 1264bp ). The reaction condition was optimized and two PCR reactions were processed in the same system. 2. Four pairs of specific oligonucleotide probes based on the mutations were designed. The specificity and reliability of the probes were verified. The results indicated that difference of Tm between different probes. was less than 2℃. The probe parameters such as GC%, Hairpin dG, Dimer dG and repeated length met the demand of design. They could hybridize under the same conditions and could distinguish two target sequences whih single base differences. 3. After checked on a nylon membrane, the selected detection probes and positive。