Combinatorial decoding: An approach for universal DNA array fabrication
Combinatorial decoding: An approach for universal DNA array fabrication
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DOI：
10.1021/ja0365577
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发表时间：
2003-11-12
影响因子：
15
通讯作者：
Walt, DR
中科院分区：
文献类型：
--
作者：
Epstein, JR;Ferguson, JA;Walt, DR
描述了一种基于光纤微球的寡核苷酸阵列，其采用连接到每个微球的寡核苷酸探针的序列作为位置标识符。每个微球用作固定阵列特征，用独特的单链寡核苷酸序列功能化并随机分布到微孔阵列中。为了确定连接到单个微球的序列，设计了一系列荧光标记的组合汇集的寡核苷酸靶溶液。每个组合解码方案旨在鉴定阵列中每个微球上特定位置处的核苷酸。通过将每个位置处的四种可能的核苷酸连接到四种不同的荧光报告染料来合成组合靶溶液。因此，当溶液与阵列杂交时，对于微球探针序列上的每个位置产生四种可能的荧光响应之一。调节杂交的严格性使得能够进行单碱基错配辨别，并且具有最高强度的信号对应于完美的核苷酸匹配。通过将阵列连续暴露于一系列组合解码池溶液，可以同时确定阵列中每个随机定位的多核苷酸官能化微球的序列。一旦映射，微球阵列可以用于任何典型的基因组微阵列实验。
A fiber optic microsphere-based oligonucleotide array is described that employs the sequence of the oligonucleotide probe attached to each microsphere as positional identifiers. Each microsphere serves as an immobilized array feature, functionalized with a unique single-stranded oligonucleotide sequence and randomly distributed into an array of microwells. To determine the sequences attached to individual microspheres, a series of fluorescently labeled combinatorial-pooled oligonucleotide target solutions was designed. Each combinatorial decoding solution is intended to identify the nucleotide at a particular position on every microsphere in the array. The combinatorial target solutions were synthesized by linking the four possible nucleotides at each position to four different fluorescent reporter dyes. As such, when the solutions were hybridized to the array, one of four possible fluorescent responses was generated for each position on a microsphere probe sequence. Adjusting the stringency of hybridization enabled single-base mismatch discrimination, and the signal with the highest intensity corresponded to the perfect nucleotide match. By consecutively exposing the array to a series of combinatorial decoding pool solutions, it was possible to simultaneously determine the sequence of every randomly positioned oligonucleotide-functionalized microsphere in the array. Once mapped, the microsphere array can be used for any typical genomic microarray experiment.