DNA DIAGNOSIS OF HEMOGLOBINOPATHIES USING MICROFABRICATED SILICON CHIPS

使用微加工硅芯片对血红蛋白病进行 DNA 诊断

• 批准号: 6325926
• 负责人: Paolo M Fortina
• 金额: $ 17.18万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325926
• 关键词: DNA; biomedical equipment development; blood disorder diagnosis; clinical biomedical equipment; diagnosis design /evaluation; gene mutation; hemoglobin Ss; hemoglobinopathy; human subject; monoclonal antibody; noninvasive diagnosis; nucleic acid sequence; oligonucleotides; polymerase chain reaction; prenatal diagnosis; sickle cell anemia; trophoblast

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.

镰状细胞病（SCD）是镰状细胞病中最常见的突变，其病因是 β-珠蛋白基因，是缬氨酸取代谷氨酸在 β链的第六个残基。这种疾病的发病率， 非洲裔美国人大约是5000名新生儿中的1名，其中约8%是非洲裔美国人。 非裔美国人为Hb S杂合子。由于SCD是一个重要的 发病率和死亡率的原因，我们建议采用硅 半导体处理技术，包括光刻、湿法 化学蚀刻和阳极键合，以开发准确，自动化和 能够直接检测正常的和/或 具有临床意义的镰状化的β S-珠蛋白等位基因 血红蛋白病由此产生的小型化DNA芯片将执行 样品制备、核酸扩增和小型化毛细管 电泳（CE）或杂交突变检测（MDBH） 用于DNA片段大小测定的高速和高通量能力， β-球蛋白基因突变检测。与此相关的是， 本申请旨在研究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。使用 在前三个月期间收集的母体外周血，胎儿 将通过不同的方法鉴定和分离滋养层细胞。 大小的硅滤波器蚀刻在硅芯片中。含单克隆抗体的微珠 对滋养层具有独特特异性和高亲和力的抗体 如果需要，将使用膜蛋白，以实现额外的 细胞分离水平。虽然这种细胞的产量低， 可以分离足够数量以允许通过PCR扩增，因此 从而能够鉴定镰状细胞基因， 广泛的非侵入性产前筛查。直接DNA扩增将 然后在微滤芯片的修改之后进行， 一部分扩增子将最终在寡核苷酸阵列上进行测试。 整合了整个β-珠蛋白重叠片段的芯片 基因预计这项技术将提高速度， 可靠性和可访问性的基础识别快速诊断 SCD变异体的复合杂合子中的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， 并允许自动化操作和集成数据采集 分析患者。