SPECIFIC DIAGNOSIS OF HEMOGLOBINOPATHIES

镰状细胞病(SCD)是人类最常见的基因突变 β-珠蛋白基因，是谷氨酸在谷氨酸的代替物 β链的第六个残基。该疾病的发病率在 非洲裔美国人大约每500名新生儿中就有1名，其中约8% 非裔美国人是HbS杂合子，因为SCD是一种重要的 发病和死亡的原因，我们建议设计一种准确的， 能够增加信心的自动化和经济高效的方法 新生儿诊断水平高发、多发病 临床上有显著的镰状血红蛋白病变。与这些相关 目标这个项目旨在建立一种等位基因特异性 荧光标记基因扩增法检测A-T 人类β-珠蛋白基因密码子6的编码序列突变。 快速非放射性方法将允许直接检测 基因组DNA中的正常或β-S珠蛋白等位基因 探针杂交或限制性内切酶切割的步骤。进阶 将开发和利用技术来筛选变种 通过开发一种策略来快速检测其他已知的 慢性阻塞性肺疾病复合杂合子中的β-珠蛋白基因突变 SCD的变种。S和S复合杂合子样本 其他已知的β基因突变将被分析为4-6个突变 通过多重反应或竞争等位基因特异性的反应 荧光标记基因扩增方法。样本： 未确定特征的突变将通过自动化诊断 基于荧光的DNA序列分析。自动电泳仪 结合实时多色荧光检测的独有 标记的引物将提供检测PCR产生的片段的手段 在一条车道上。最后，需要调查的是一项 胎儿血气分析无创产前诊断方法 从母体外周血中获取滋养层细胞。胎儿 孕期孕妇外周血中的滋养层细胞 用独有的单抗鉴定和分离 针对滋养细胞膜蛋白的特异性和高亲和力。它 预计胎儿滋养层细胞可以在胚胎发育过程中分离出来 怀孕前三个月。虽然这种电池的产量很低，但一种 可以分离出足够的数量以允许通过PCR进行扩增，因此 使得能够识别镰状细胞基因，并最终允许 广泛的无创性产前筛查。

The cause of sickle cell disease (SCD), the most common mutation in the beta-globin gene, is the substitution of valine for glutamic acid at the sixth residue of the beta chain. The incidence of the disorder among African-Americans is approximately 1 in 500 births, with about 8% of African-Americans being heterozygous for Hb S. Since SCD is a significant cause of morbidity and mortality, we propose to devise an accurate, automated and cost-effective methods capable of increasing the confidence level of neonatal diagnosis of frequently-encountered, clinically-significant sickling hemoglobinopathies. Related to these goals this project aims to establish an allele-specific fluorescence-tagged gene amplification protocol to assay for the A to T mutation in the sequence encoding codon 6 of the human beta-globin gene. The rapid nonradioactive approach will allow direct detection of the normal or the beta-s-globin allele in genomic DNA without the additional steps of probe hybridization or restriction enzyme cleavage. Advanced techniques will be developed and utilize for screening for variants of SCD by developing a strategy for rapidly detecting additional known mutations within the beta-globin gene in compound heterozygotes for variants of SCD. Samples from compound heterozygotes for beta-s and other known beta-gene mutations will be analyzed for 4-6 mutations per reaction by multiplex or a competitive allele-specific fluorescence-tagged gene amplification protocol. Samples with uncharacterized mutations will be diagnosed by automated fluorescence-based DNA sequence analysis. Automated electrophoresis combined with real time multicolor fluorescence detection of unique labeled primers will provide the means to detect PCR-generated fragments in a single lane. Finally, to be investigated is the feasibility of a non-invasive method of prenatal diagnosis by analysis of fetal trophoblast cells obtained from maternal peripheral blood. Fetal trophoblast cells from maternal peripheral blood during pregnancy will be identified and isolated by means of monoclonal antibodies of unique specificity and high affinity against trophoblast membrane proteins. It is anticipated that fetal trophoblast cells can be isolated during the first trimester of pregnancy. Although the yield of such cells is low, a sufficient number can be isolated to allow amplification by PCR, thus enabling identification of the sickle cell gene and ultimately allowing widespread noninvasive prenatal screening.