Yim Seong-Chun, Park Hyun Gyu, Chang Ho Nam, Cho Dae-Yeon
Department of Chemical and Biomolecular Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Anal Biochem. 2005 Feb 15;337(2):332-7. doi: 10.1016/j.ab.2004.11.034.
We describe here an efficient microarray-based multiplex assay to detect Korean-specific mutations in breast cancer susceptibility gene BRCA1 using direct probe/target hybridization. Allele-specific oligonucleotides were covalently immobilized on an aldehyde-activated glass slide to prepare an oligonucleotide chip. From a wild-type sample, a two-step method was used to generate labeled multiplex polymerase chain reaction (PCR) amplification products of genomic regions containing the mutation sites. Amino allyl-dUTP, an amine-modified nucleotide, was incorporated during multiplex PCR amplifications and a monofunctional form of cyanine 3 dye was subsequently attached to the reactive amine group of the PCR products. Hybridization of the labeled PCR products to the oligonucleotide chip successfully identified all of the genotypes for the selected mutation sites. This work demonstrates that oligonucleotides chip-based analysis is a good candidate for efficient clinical testing for BRCA1 mutations when combined with the indirect strategy to prepare labeled target samples.
我们在此描述一种基于微阵列的高效多重检测方法,该方法利用直接探针/靶标杂交来检测乳腺癌易感基因BRCA1中的韩国特异性突变。等位基因特异性寡核苷酸共价固定在醛基活化的载玻片上以制备寡核苷酸芯片。对于野生型样本,采用两步法生成包含突变位点的基因组区域的标记多重聚合酶链反应(PCR)扩增产物。在多重PCR扩增过程中掺入氨基烯丙基-dUTP(一种胺修饰的核苷酸),随后将单功能形式的花青3染料连接到PCR产物的反应性胺基上。标记的PCR产物与寡核苷酸芯片的杂交成功鉴定了所选突变位点的所有基因型。这项工作表明,当与制备标记靶标样本的间接策略相结合时,基于寡核苷酸芯片的分析是BRCA1突变高效临床检测的良好候选方法。