DNA DIAGNOSIS OF HEMOGLOBINOPATHIES USING MICROFABRICATED SILICON CHIPS

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5000名新生儿中就有一名，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，我们建议采用硅 半导体加工技术，包括湿法光刻 化学蚀刻和阳极键合，以开发精确、自动化和 经济实惠的微型设备，能够直接检测正常和/或 临床上有意义的镰刀样病变的β-珠蛋白等位基因 血红蛋白病。由此产生的微型DNA芯片将执行 样品制备、核酸扩增和微型化毛细管 电泳法(CE)或杂交法(MDBH) 用于DNA片段大小调整和处理的高速和吞吐量能力 β-珠蛋白基因突变检测。与这一目标相关的， 应用目的是研究一种非侵入性方法的可行性 通过分析胎儿滋养层细胞进行产前诊断。vbl.使用 妊娠前三个月采集的孕妇外周血，胎儿 滋养层细胞将通过不同的方法进行鉴定和分离 在硅片上蚀刻的大小硅过滤器。含有单抗的珠子 具有独特特异性和高亲和力的滋养细胞抗体 如果有必要，将使用膜蛋白来实现额外的 细胞分离的水平。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。直接DNA扩增将 然后在修改微滤芯片之后执行，并且 部分扩增产物最终将在寡聚阵列上进行测试 整合了整个β-珠蛋白重叠片段的芯片 吉恩。预计这项技术将提高速度， 碱基识别的可靠性和可获得性，用于快速诊断 SCD变异型复合杂合子中的β-珠蛋白基因突变。 最后，半导体芯片制造技术的实现 将减少污染、样品和试剂的消耗，增加 吞吐量，并允许自动化操作和集成数据采集 并对患者进行分析。

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 5000 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 adapt silicon semiconductor processing techniques including photolithography, wet chemical etching and anodic bonding, to develop accurate, automated and cost-effective microdevices capable of direct detection of normal and/or betaS-globin alleles for clinically-significant sickling hemoglobinopathies. The resulting miniaturized DNA chip will perform sample preparation, nucleic acid amplification and miniaturized capillary electrophoresis (CE) or mutation detection by hybridization (MDBH) with high speed and throughput capabilities for DNA fragment sizing and mutation detection in the beta-globin gene. Related to this goal, the application aims to investigate the feasibility the a non-invasive method of prenatal diagnosis by analysis of fetal trophoblast cells. Using maternal peripheral blood collected during the first trimester, fetal trophoblast cells will be identified and isolated by means of different sized silicon filters etched in silicon chips. Beads containing monoclonal antibodies of unique specificity and high affinity against trophoblast membrane proteins will be employed, if necessary to achieve an additional level of cell separation. 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 non-invasive prenatal screening. Direct DNA amplification will then be performed following modification of the microfiltration chip and a fraction of the amplicons will be finally tested on an oligo-arrayed chip which incorporates overlapping segments of the whole beta-globin gene. It is anticipated that this technology will improve the speed, reliability, and accessibility of base recognition for rapid diagnosis of beta-globin gene mutations in compound heterozygotes for variants of SCD. Finally, implementation of the semiconductor chip manufacture technology will reduce contamination, consumption of sample and reagents, increase throughput and allow automated operation and integrated data acquisition and analysis of patients.