【作者】 杨谷；

【导师】 朱斌；

【作者基本信息】 苏州大学 ， 遗传学， 2010， 硕士

【摘要】 目的:建立稳定的单碱基延伸结合荧光光谱分析鉴别SNP中2种碱基比例的方法,并应用于羊水标本的检测,探讨该技术产前诊断唐氏综合征的可行性,为最终建立一种简便、可靠的非创伤性诊断唐氏综合征技术打下基础。方法:选取一个胎盘源性表达的基因PLAC4,利用分子“开/关”技术对其中一个SNP位点rs8130833(A和G)进行特异性扩增,最后采用荧光分光光度计对扩增产物进行光谱分析。在方法稳定的基础上,用于63例唐氏筛查高危孕妇的羊水标本检测,获得诊断唐氏胎儿的置信区间。结果:在方法建立时,模板中A:G比例分别按1:1,1:2,2:1混匀,扩增后检测产物,A:G荧光光谱强度比例与原反应模板一致,表明我们建立的鉴别SNP等位位点比例的方法是可行的。63例羊水标本,经分子“开/关”鉴定,33例为杂合,进一步检测A:G比例,获得置信区间结果为正常胎儿为0.75-1.29,唐氏患儿(AAG)和(AGG)分别为1.85-2.29和0.39-0.67,与染色体核型分析的对比,诊断特异性达94.1%。结论:本研究中建立的SNP等位位点比例法产前诊断唐氏综合征简便,直观,特异性强。可进一步优化、完善,用于疾病的非创伤性产前诊断。更多还原

【Abstract】 Objective: To set up a simple, reliable non-invasive prenatal diagnosis technical for Down’s syndrome eventually, a stable method, which combined single base extension with fluorescence spectrometry, was established to identify SNP allelic ratios, and amniotic fluid samples were used to explore the feasibility of this method for prenatal diagnosing Down’s syndrome.Methods: A placenta derived expression gene PLAC4 was selected, a SNP loci rs8130833 (A and G) in PLAC4 was amplified by on/off switch to eliminate non-specific amplification. Finally amplified products were analyzed by fluorescence spectrophotometer. On the basis of the method, 63 amniotic fluid samples obtained from Down’s high risk pregnant women were detected to get confidence intervals for diagnosing Down’s syndrome.Results: When established the method, template’s A:G ratio were mixed as 1:1,1:2,2:1 respectively. After amplification, the products were detected. The A:G ratio of fluorescence intensity was consistent with the ratio of original template, which indicated that the method we established to identify SNP allelic ratio was feasible. Identified by on/off switch, 33 cases were heterozygous in 66 cases of amniotic fluid samples. Through detecting A:G ratio of heterozygous samples, the confidence intervals were obtained, the euploid was 0.75-1.29, Down’s samples(AAG or AGG) were 1.85-2.29 or 0.39-0.67. Compared with karyotype analysis, the specificity was 94.1%.Conclusion: The method, which we established to identify SNP allelic ratios, was simple, direct and specific, and could be used to diagnose Down’s syndrome. Further optimized, it could be applied for non-invasive prenatal diagnosis.更多还原