Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer
Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer
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DOI：
10.1038/86730
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发表时间：
2001-04-01
影响因子：
46.9
通讯作者：
Linsley, PS
中科院分区：
文献类型：
--
作者：
Hughes, TR;Mao, M;Linsley, PS
我们描述了一个灵活的基因表达分析系统，使用成千上万的寡核苷酸阵列，通过喷墨打印方法，采用标准的酰胺磷化学原位合成。我们描述了杂交特异性和敏感性对参数的依赖性，包括寡核苷酸长度、杂交严格性、序列一致性、样品丰度和样品制备方法。我们发现60聚寡核苷酸可以可靠地检测复杂生物样品中每个细胞一个拷贝的转录本比率，并且喷墨阵列与几种不同的样品扩增和标记技术兼容。此外，仅使用单个精心选择的每个基因寡核苷酸的结果与使用互补DNA （cDNA）阵列获得的结果密切相关。测量结果不同的大多数基因是基因家族的成员，只能通过寡核苷酸来区分。由于每个阵列可以指定不同的寡核苷酸序列，我们预计喷墨寡核苷酸阵列技术将在各种DNA微阵列应用中发挥重要作用。
We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.