HLA-B寡核苷酸分型芯片制备方法的建立及初步应用

发布时间：2018-06-05 03:37

本文选题：寡核苷酸芯片 + 分型探针　；参考：《天津医科大学》2006年硕士论文

【摘要】：寡核苷酸芯片(oligonucleotide chip)技术近年来发展迅速,其制备方法随着应用目的不同而多种多样,其中芯片片基的表面处理是制备芯片的关键技术之一。本实验用丙基三甲氧基硅烷(3-glycidoxypropyltrimethoxysilane)作为芯片片基表面处理剂自行制备了环氧硅烷寡核苷酸芯片,并设计分型探针用于HLA-B抗原的基因分型。芯片片基首先在清洗液中用超声清洗干净,100mmol/L NaOH处理使其表面产生自由羟基,然后用丙基三甲氧基硅烷作为硅烷偶联剂处理玻片表面,修饰后的玻片能共价结合氨基修饰的寡核苷酸分型探针。探针5'末端经过氨基修饰后,用芯片点样仪按照预先设计的编号位置在玻片上形成规则矩阵。点样后的玻片经过水合、洗脱液处理、烘烤固定以后可用于杂交。寡核苷酸芯片技术以其快速、高通量、大信息量检测等特点而应用广泛,尤其在对HLA-B分型方面显出很大优势。HLA系统是调控人体特异性免疫应答和决定疾病易感性个体差异的主要基因系统,已经发现有上百种疾病的发生发展与HLA-B有关,包括遗传与环境共同起作用的疾病,因此HLA-B的多态性检测在科研和医学实践中具有重要意义。由于HLA具有众多的等位基因及其高度多态性,传统血清学和细胞学分型方法对HLA分型存在判断误差问题,尤其是HLA Ⅰ类抗原分型。我们根据中国北方汉族人群HLA-B常见基因位点及临床分型分辨度特征,同时考虑强直性脊柱炎、幼年型糖尿病等与HLA密切相关的遗传病等因素,设计了66条特异性寡核苷酸中分辨度分型探针,用于HLA-B基因分型。采用TKM法自人血细胞中提取基因组DNA,并以之为模板,Cy3荧光标
[Abstract]:The technology of oligonucleotide chip-based oligonucleotide has been developed rapidly in recent years, and its preparation methods vary with the purpose of application. The surface treatment of chip is one of the key technologies in the fabrication of chips. An epoxysilane oligonucleotide chip was prepared by using propyl trimethoxysilane 3-glycidoxypropyltrimethoxysilaneas as the surface treatment agent. The microarray chip was first cleaned by ultrasound to clean the surface of 100 mmol / L NaOH and then treated with propyl trimethoxysilane as silane coupling agent to treat the glass surface. The modified slides can covalently bind to amino modified oligonucleotide typing probes. The probe 5'end was modified with amino, and a regular matrix was formed on the glass by using the chip spotting apparatus according to the pre-designed numbering position. After hydration, eluent treatment, baking and fixation, the glass slides can be used for hybridization. Oligonucleotide chip technology is widely used for its rapid, high throughput, large information detection and other characteristics. Especially in HLA-B typing, HLA system is the main gene system to regulate specific immune response and determine individual differences in disease susceptibility. It has been found that the occurrence and development of hundreds of diseases are related to HLA-B. Therefore, the detection of HLA-B polymorphism plays an important role in scientific research and medical practice. Because HLA has many alleles and high polymorphism, traditional serological and cytological typing methods have some problems in judging HLA typing, especially HLA class I antigen typing. According to the characteristics of HLA-B common gene loci and clinical typing resolution in the Han population of northern China, we also considered the factors such as ankylosing spondylitis, juvenile diabetes mellitus and other genetic diseases closely related to HLA. 66 specific oligonucleotide resolution typing probes were designed for HLA-B genotyping. Genomic DNA was extracted from human blood cells by TKM method and used as a template for Cy3 fluorescence labeling.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346;Q789

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