Rapid Cystic Fibrosis DNA Mutation Screening Test

囊性纤维化 DNA 突变快速筛查试验

• 批准号: 6689680
• 负责人: WLODEK MANDECKI
• 金额: $ 10万
• 依托单位: PHARMASEQ, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6689680
• 关键词: charge coupled device camera; chloride channels; cystic fibrosis; diagnosis design /evaluation; diagnostic tests; fluorescence microscopy; fluorescent dye /probe; gene expression; gene mutation; genetic screening; genotype; high throughput technology; inborn biological transport disorder; inborn metabolism disorder diagnosis; nucleic acid hybridization; oligonucleotides; polymerase chain reaction; rapid diagnosis; technology /technique development

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is a highly morbid, autosomal recessive disease caused by one or more mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The CFTR protein is important in transporting sodium and chloride ions through the membranes of epithelial cells lining the lungs and other organs. In Caucasians, CF is the most lethal inherited disease of childhood (frequency of 1:3,300). In the U.S., about 25,000 people have CF and about 850 individuals are diagnosed with CF each year. CF affects all races and ethnic groups and it is underdiagnosed, especially in minorities. Present genetic screening assays are best suited for detecting CF in Caucasians and Ashkenazi Jews but, the mutations currently assayed for in the CF screening does not detect mutations specific for Black Americans and Hispanics. Electronic microtransponders will be used in a novel DNA detection system to quickly and accurately detect large numbers of CF DNA mutations in a single high-speed flow assay. Each microtransponder is an integrated circuit composed of photocells, memory and antenna. It stores information identifying the sequence of an attached oligonucleotide probe in its electronic memory. Complementary, dye-tagged target DNA sequences bind to probes on microtransponders and the results are read in a high-speed flow reader. The Specific Aims of Phase I are to prove feasibility of a novel, rapid and inexpensive microtransponder-based CFTR screening assay: 1) Select ten important cystic fibrosis (CF) mutations in Caucasians, Black Americans and Hispanics and prepare synthetic DNA probes to these mutations; 2) Prepare primers for all CFTR mutations tested for use in multiplex PCR reaction; 3) Covalently attach optimized DNA probes to microtransponders, perform a microtransponder-based DNA probe assay using cell lines containing CFTR gene mutations and match results to purchased cell line descriptions; 4) Perform the microtransponder CF assay on a small number of patient and control samples and compare results to standard methods used in a genetics laboratory in a major medical center in New Jersey. The microtransponder-based, multiplex assay proposed with extended mutation detection for domestic ethnic groups will significantly increase the utility of this important assay for more U.S. patients by adding important CFTR mutations observed in Blacks and Hispanics into a single, affordable, accurate, high-speed assay.

描述（由申请人提供）：囊性纤维化（CF）是一种高度发病的常染色体隐性遗传疾病，由编码囊性纤维化跨膜电导调节（CFTR）蛋白的基因中的一个或多个突变引起。 CFTR 蛋白对于通过肺和其他器官内壁上皮细胞膜转运钠离子和氯离子非常重要。在白种人中，CF 是儿童时期最致命的遗传性疾病（频率为 1:3,300）。在美国，大约有 25,000 人患有 CF，每年约有 850 人被诊断患有 CF。 CF 影响所有种族和族裔群体，但诊断不足，尤其是少数族裔。目前的基因筛查测定最适合检测白种人和德系犹太人的 CF，但是目前在 CF 筛查中测定的突变并不能检测到美国黑人和西班牙裔的特异性突变。电子微转发器将用于新型 DNA 检测系统，可在单次高速流动测定中快速准确地检测大量 CF DNA 突变。每个微应答器都是由光电管、存储器和天线组成的集成电路。它将识别所连接的寡核苷酸探针的序列的信息存储在其电子存储器中。互补的、染料标记的目标 DNA 序列与微应答器上的探针结合，并在高速流阅读器中读取结果。第一阶段的具体目标是证明一种新颖、快速且廉价的基于微应答器的 CFTR 筛查测定的可行性：1) 选择白种人、美国黑人和西班牙人中的 10 个重要的囊性纤维化 (CF) 突变，并针对这些突变制备合成 DNA 探针; 2) 为所有测试的CFTR突变制备引物，用于多重PCR反应； 3) 将优化的 DNA 探针共价连接到微应答器上，使用含有 CFTR 基因突变的细胞系进行基于微应答器的 DNA 探针测定，并将结果与​​购买的细胞系描述进行匹配； 4) 对少量患者和对照样本进行微应答器 CF 测定，并将结果与​​新泽西州主要医疗中心遗传学实验室使用的标准方法进行比较。基于微应答器的多重检测提出了针对国内种族群体的扩展突变检测，通过将在黑人和西班牙裔中观察到的重要 CFTR 突变添加到单一、经济实惠、准确、高速的检测中，将显着提高这一重要检测对更多美国患者的实用性。化验。