本文選題：寡核苷酸芯片 + 分型探針　； 參考：《天津醫(yī)科大學》2006年碩士論文

【摘要】：寡核苷酸芯片(oligonucleotide chip)技術(shù)近年來發(fā)展迅速,其制備方法隨著應(yīng)用目的不同而多種多樣,其中芯片片基的表面處理是制備芯片的關(guān)鍵技術(shù)之一。本實驗用丙基三甲氧基硅烷(3-glycidoxypropyltrimethoxysilane)作為芯片片基表面處理劑自行制備了環(huán)氧硅烷寡核苷酸芯片,并設(shè)計分型探針用于HLA-B抗原的基因分型。 芯片片基首先在清洗液中用超聲清洗干凈,100mmol/L NaOH處理使其表面產(chǎn)生自由羥基,然后用丙基三甲氧基硅烷作為硅烷偶聯(lián)劑處理玻片表面,修飾后的玻片能共價結(jié)合氨基修飾的寡核苷酸分型探針。探針5'末端經(jīng)過氨基修飾后,用芯片點樣儀按照預(yù)先設(shè)計的編號位置在玻片上形成規(guī)則矩陣。點樣后的玻片經(jīng)過水合、洗脫液處理、烘烤固定以后可用于雜交。 寡核苷酸芯片技術(shù)以其快速、高通量、大信息量檢測等特點而應(yīng)用廣泛,尤其在對HLA-B分型方面顯出很大優(yōu)勢。HLA系統(tǒng)是調(diào)控人體特異性免疫應(yīng)答和決定疾病易感性個體差異的主要基因系統(tǒng),已經(jīng)發(fā)現(xiàn)有上百種疾病的發(fā)生發(fā)展與HLA-B有關(guān),包括遺傳與環(huán)境共同起作用的疾病,因此HLA-B的多態(tài)性檢測在科研和醫(yī)學實踐中具有重要意義。由于HLA具有眾多的等位基因及其高度多態(tài)性,傳統(tǒng)血清學和細胞學分型方法對HLA分型存在判斷誤差問題,尤其是HLA Ⅰ類抗原分型。我們根據(jù)中國北方漢族人群HLA-B常見基因位點及臨床分型分辨度特征,同時考慮強直性脊柱炎、幼年型糖尿病等與HLA密切相關(guān)的遺傳病等因素,設(shè)計了66條特異性寡核苷酸中分辨度分型探針,用于HLA-B基因分型。 采用TKM法自人血細胞中提取基因組DNA,并以之為模板,Cy3熒光標
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346;Q789

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本文編號：1980318