Universal DNA microarray method for multiplex detection of low abundance point mutations
Universal DNA microarray method for multiplex detection of low abundance point mutations
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DOI：
10.1006/jmbi.1999.3063
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发表时间：
1999-09-17
影响因子：
5.6
通讯作者：
Barany, F
中科院分区：
文献类型：
--
作者：
Gerry, NP;Witowski, NE;Barany, F
癌症是由调节细胞生长和分化的基因中的多个突变的积累引起的。在许多基因中鉴定这样的突变代表了遗传分析中的重大挑战，特别是当肿瘤样品中的大多数DNA来自野生型基质时。为了克服这些困难，我们开发了一种新型的DNA微芯片，结合聚合酶链反应/连接酶检测反应（PCR/LDR）与“邮编”杂交。当且仅当存在突变时，适当设计的等位基因特异性LDR引物与相邻的荧光标记引物共价连接。等位基因特异性LDR引物在其5 '端含有"邮政编码互补物"，其用于将LDR产物引导至共价连接至三维凝胶基质阵列的特定邮政编码地址。由于邮政编码与靶序列或基因组中的其他序列都没有同源性，因此检测不到由于错配杂交而产生的假信号。邮政编码序列保持不变，其互补序列可以附加到任何一组LDR引物上，使我们的邮政编码阵列具有通用性。使用K-ras基因作为模型系统，多重PCR/LDR，然后与原型3 × 3邮政编码阵列杂交，正确鉴定了肿瘤和细胞系DNA中的所有突变。突变存在于野生型DNA水平的不到百分之一可以区分。通用阵列可用于快速检测任何目标基因中的低丰度突变。(C)北京：科学出版社.
Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents;I significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wildtype stroma.:To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can Ire appended to any set of LDR primers, making our zip-code arrays universal. Using the K-ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3 x 3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest. (C) 1999 Academic Press.