Rapid Cystic Fibrosis DNA Mutation Screening Test

描述（由申请人提供）：囊性纤维化（CF）是一种高度病态的常染色体隐性遗传病，由编码囊性纤维化跨膜传导调节因子（CFTR）蛋白的基因发生一个或多个突变引起。CFTR蛋白在通过肺和其他器官的上皮细胞膜运输钠离子和氯离子方面很重要。在白种人中，CF是儿童时期最致命的遗传性疾病（发病率为1:3 300）。在美国，大约有25000人患有CF，每年大约有850人被诊断为CF。CF影响所有种族和族裔群体，但未得到充分诊断，尤其是少数民族。目前的基因筛查方法最适合于检测白种人和德系犹太人的CF，但是，目前在CF筛查中检测的突变并不能检测到美国黑人和西班牙裔美国人的特异性突变。电子微转发器将用于一种新的DNA检测系统，在一次高速流动试验中快速准确地检测大量CF DNA突变。每个微应答器是由光电池、存储器和天线组成的集成电路。它在其电子存储器中存储识别附着的寡核苷酸探针序列的信息。互补的，染料标记的目标DNA序列与微转发器上的探针结合，结果在高速流读取器中读取。第一阶段的具体目标是证明一种新型、快速和廉价的基于微应答器的CFTR筛选方法的可行性：1)在高加索人、美国黑人和西班牙裔人中选择10个重要的囊性纤维化（CF）突变，并制备这些突变的合成DNA探针；2)准备所有CFTR突变的引物，用于多重PCR反应；3)将优化的DNA探针共价连接到微转发器上，使用含有CFTR基因突变的细胞系进行基于微转发器的DNA探针检测，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分析，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方法将扩展国内少数民族的突变检测，通过将在黑人和西班牙裔人群中观察到的重要CFTR突变添加到一个单一的、负担得起的、准确的、高速的检测方法中，将显著提高这一重要检测方法对更多美国患者的实用性。

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.