Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2
Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2
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DOI：
10.1038/75452
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发表时间：
2000-05-01
影响因子：
46.9
通讯作者：
Barany, F
中科院分区：
文献类型：
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作者：
Favis, R;Day, JP;Barany, F
基于阵列的突变检测方法通常依赖于荧光标记的查询序列与表面结合的寡核苷酸探针的直接杂交。这些探针含有小的序列变异或完全匹配的序列。与每个寡核苷酸探针结合的荧光强度旨在揭示哪个序列与查询序列完全互补（1）。然而，这些方法并不总是成功的，特别是对于小的移码突变的检测。在这里，我们描述了一种多重检测，通过使用改良的PCR均匀扩增每个扩增子（PCR/PCR）（2），然后进行连接酶检测反应（LDR）（3）来检测小的插入和缺失。通过用通用DNA微阵列筛选反应产物来鉴定突变（4），该微阵列将突变检测与阵列杂交分离并提供高灵敏度。使用德系犹太人群体中的三种BRCA 1和BRCA 2创始突变（BRCA 1 185 delAG; BRCA 1 5382 insC; BRCA 2 6174 delT）（5）作为模型系统，该检测试剂盒在多重反应中很容易检测到这些突变。我们的研究结果表明，通用的PCR/PCR/LDR 2产品的微阵列分析允许快速识别小的插入和缺失突变的背景下，在临床诊断和人口研究。
Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence(1). However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR)(2), followed by ligase detection reaction (LDR)(3). Mutations were identified by screening reaction products with a universal DNA microarray(4), which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT)(5) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR2 products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.