【作者】 易萍；

【导师】 李力；

【作者基本信息】 第三军医大学 ， 妇产科学， 2006， 博士

【摘要】 背景与目的:近年来研究发现在妊娠过程中母体血浆中存在游离胎儿DNA,这一发现为基于母体外周血的无创性产前诊断开辟了新的领域。胎儿DNA一半来自于母亲,另一半来自于父亲,目前已报道利用母体血浆DNA来检测胎儿父系来源的致病基因,可应用于父亲为常染色体显性遗传病患者的胎儿无创性产前诊断,而且将这种检测方法应用于父亲为常染色体隐性遗传病携带者的产前诊断,也可使50%的胎儿避免了传统的绒毛吸取或羊膜腔穿刺检查。但由于母体血浆总DNA量少,且在血浆总DNA中胎儿DNA所占的比例甚微,母体等位DNA片段可干扰胎儿DNA的检测,因此对这种低丰度胎儿DNA检测必须采用高灵敏度和高特异性的分析系统。点突变或单核苷酸多态性(single nucleotide polymorphism,SNP)的检测是目前DNA分子诊断中的主要研究内容,对母体血浆中的这些单核苷酸差异的检测在技术上要求更高,目前所用的检测方法仍不够理想。DNA连接酶对单个核苷酸差异具有很强的鉴别能力,其核心在于它可以把与模板完全配对的两相邻寡核苷酸片段连接起来,如果连接处出现一个碱基错配,连接反应就不能进行。以该酶为基础的技术如空隙-连接酶链反应(gap ligase chain reaction,Gap-LCR)和连接酶检测反应(ligase detection reaction,LDR)在低丰度基因单核苷酸差异检测方面具有很强的应用潜力。因此本研究拟以β地中海贫血的常见突变和雌激素受体α的SNP位点(c.454-397T>C)作为研究对象,在Gap-LCR或LDR技术的基础上,结合荧光定量PCR、毛细管电泳和纳米金新型通用型芯片来检测低丰度基因的单核苷酸差异,探索检测母体血浆中胎儿父系来源DNA点突变的新方法,为胎儿遗传性疾病的无创性产前诊断寻求新的有效途径。主要方法与结果如下:第一部分:母体血浆DNA实验模型的建立1.用反向斑点杂交技术检测β地中海贫血IVS-Ⅱ-654(C→T)、CD17(A→T)、CD26(G→A)、CD41-42(-CTTT)和CD71-72(+A)突变的外周血标本,然后进行DNA序列分析,结果发现反向斑点杂交技术在突变位点周围存在SNP时可造成误诊。2.利用pVUⅡ限制酶酶切方法鉴定雌激素受体α基因SNP(c.454-397T>C)分型,更多还原

【Abstract】 Background and purposes: Free fetal DNA has been found in maternal plasma recently, which facilitates the development of noninvasive prenatal diagnostic approaches based simply on the analysis of a maternal blood sample.The detection of the paternally transmitted pathogenic allele in maternal plasma has been reported to be applicable to the prenatal diagnosis of paternally inherited autosomal dominant traits. If a similar approach is used for the prenatal assessment of autosomal recessive conditions, an invasive prenatal diagnostic procedure would be avoided in 50% of these pregnancies, in whom the lack of inheritance of the paternal mutation by the fetus is confirmed by maternal plasma analysis. However, the low quantity of fetal DNA in the maternal circulation and interference from an excessive amount of maternal DNA makes detection of fetal mutations difficult. The diagnostic reliability depends on the absolute sensitivity and specificity of the assay system. Present clinical DNA diagnosis mainly concentrates on detection of point mutation and single-nucleotide polymorphism (SNP). The technique detecting the discrimination of single-nucleotide differences between fetal DNA and maternal DNA is challenging and demands the adoption of more highly sensitive and specific analytical systems. However, reported diagnostic procedures are still beyond our satisfaction. DNA ligase has the capacity to accurately discriminate a single nucleotide polymorphism. Two adjacent oligonucleotide primers hybridized to a single strand of target DNA will be ligated by ligase only if there is an exact match to the target sequence. Ligation will not occur in the presence of a mismatch. The fidelity of DNA ligases proviedes the basis for developing ligase-based technologies to highly sensitively detect low abundance point mutations. Gap-gap ligase chain reaction (gap-LCR) and ligase detection reaction (LDR) achieve both signal detection and amplification through repeated strand reactions catalyzed by DNA ligase. The aim of the study is to detect low abundance point mutations by using Gap-LCR and real-time PCR and更多还原