Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms
Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms
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DOI：
10.1016/s1525-1578(10)60531-4
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发表时间：
2004-11-01
影响因子：
4.1
通讯作者：
WalkerPeach, CR
中科院分区：
文献类型：
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作者：
Hadd, AG;Laosinchai-Wolf, W;WalkerPeach, CR
开发用于检测与囊性纤维化（CF）相关的常见基因突变的简单快速方法需要获得阳性对照样品，包括内含子8的5/7/9 T变体。我们使用PCR和一个简单的多重珠阵列检测，以确定5/7/9 T控制样品从29个市售的DNA样品。将未纯化的PCR产物直接与含有等位基因特异性捕获探针的颜色编码珠杂交，用于5/7/9 T检测。使用反向互补寡核苷酸、单个PCR产物和多重PCR产物在复杂CFTR筛选测定中进行5/7/9 T检测，研究了测定的性能。通过对来自每个捕获探针的相对信号强度进行分组，对样品进行基因分型。在29份市售DNA样本中，共检测到5 T/7 T 2例，5 T/9 T 2例，7 T/9 T 9例，7 T/7 T 11例，9 T/9 T 5例。通过DNA测序确认每个样品组内的基因型。该测定与从全血中分离的10至1000 ng基因组DNA的分析相容，并允许从反射变体中单独鉴定主要CFTR突变。阳性对照的正确鉴定证明了简单微珠阵列测定的实用性，并为临床实验室的测定优化和常规质量控制提供了可获得的样品。
The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.