Pease A C, Solas D, Sullivan E J, Cronin M T, Holmes C P, Fodor S P
Affymetrix, Santa Clara, CA 95051.
Proc Natl Acad Sci U S A. 1994 May 24;91(11):5022-6. doi: 10.1073/pnas.91.11.5022.
In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.
在分子生物学的许多领域，都需要快速提取和分析遗传信息；然而，当前用于DNA序列分析的技术速度慢且劳动强度大。我们在此报告了如何利用现代光刻技术，通过生成密集排列的寡核苷酸探针的微型阵列来促进序列分析。这些探针阵列，即DNA芯片，随后可应用于并行DNA杂交分析，直接产生序列信息。在一个初步实验中，通过16个化学反应循环制备了一个1.28×1.28厘米的包含256种不同八核苷酸的阵列，这一过程需要4小时完成。然后通过落射荧光显微镜检测荧光标记的寡核苷酸靶标的杂交模式。来自互补探针的荧光信号比单碱基对或双碱基对杂交错配的信号强5至35倍，证明了在识别互补序列方面的特异性。该方法应被证明是人类遗传学和诊断、病原体检测以及DNA分子识别快速研究的有力工具。
