In situ synthesis of oligonucleotide arrays by using soft lithography
In situ synthesis of oligonucleotide arrays by using soft lithography
复制标题
使用软光刻原位合成寡核苷酸阵列
DOI：
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Z. H. Lu
中科院分区：
文献类型：
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作者：
P. Xiao;N. He;Z. Liu;Q. He;X. Sun;Z. H. Lu
本文介绍了利用软光刻技术在玻璃表面原位合成寡核苷酸阵列。在该方法中，基于标准亚磷酰胺化学方案，通过用一组聚二甲基硅氧烷（PDMS）微印模印刷载玻片来实现偶联，在所述微印模上涂布核苷单体和四唑混合溶液。PDMS的弹性特性允许其在印刷耦合中与载玻片进行保形接触。关于印刷偶联的效率，比较了直接滴加偶联和接触偶联合成的20-mer寡核苷酸的杂交显微镜图像，两种方法的荧光强度没有显示出显著差异。通过末端标记荧光核苷酸法研究了偶联效率，得到了97%的分步产率。研制了一种高质量、高空间分辨率、大尺寸的PDMS微印模，该微印模在一个玻璃基片上集成了168个不同的微印模，用于合成寡核苷酸阵列。通过等离子体放电处理对印模进行改性以改善其表面润湿性，从而使微印模可用于寡核苷酸阵列的制备。为了改善载玻片与不同微压印的接触效果，研制了一种移动式压印头。在印刷耦合中使用较高沸点的溶剂以抑制溶剂挥发并保持微印模的不同特征上的试剂的一致性。采用一种有效的方法消除含有羟基的小分子芯片上残留的反应性核苷。制备了与靶序列匹配和错配的四种探针的特异性寡核苷酸阵列，以鉴定完全匹配和错配序列。
This paper describes the in situ synthesis of oligonucleotide arrays on glass surfaces by using soft lithography. In this method, based on the standard phosphoramidite chemistry protocol, the coupling was achieved by the glass slide being printed with a set of polydimethylsiloxane (PDMS) microstamps, on which was spread nucleoside monomer and tetrazole mixed solution. The elastic characteristic of PDMS allowed it to make conformal contact with the glass slide in the printing coupling. With regard to the efficiency of the printing coupling, the hybridization microscope images of 20-mer oligonucleotide synthesized via the directly drip-dropped coupling and the contact coupling were compared; the fluorescence intensities of the two methods showed no significant differences. The coupling efficiency was also investigated via an end-labelled fluorescence nucleotide method and a stepwise yield of 97% was obtained. A high-quality, high spatial resolution and large-scale PDMS stamp, which was developed by integrating 168 different microstamps on one glass substrate for synthesizing oligonucleotide arrays. The stamp was modified to improve the surface wettability by plasma discharge treatments, so that microstamps could be used to fabricate oligonucleotide arrays. A motional printing head was developed to improve the contact effect of the glass slide with different microstamps. A higher boiling point solvent was used in the printing coupling to inhibit solvent volatilization and to maintain the consistency of reagents on different features of the microstamp. An effective method was used to eliminate residual reactive nucleosides on chips with small molecules containing a hydroxyl group. A specific oligonucleotide array of four probes both matched and mismatched with the target sequence was fabricated to identify the perfect match and mismatch sequences.
DOI：
10.1073/pnas.94.6.2150
发表时间：
1997-03-18
影响因子：
11.1
作者：
Heller, RA;Schena, M;Davis, RW
通讯作者：
Davis, RW