A picoliter chamber array for cell-free protein synthesis
A picoliter chamber array for cell-free protein synthesis
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DOI：
10.1093/jb/mvh102
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发表时间：
2004-08-01
影响因子：
2.7
通讯作者：
Tamiya, E
中科院分区：
文献类型：
--
作者：
Kinpara, T;Mizuno, R;Tamiya, E
人类基因组测序的完成，将研究的重点从基因转移到了蛋白质。在这方面，蛋白质库芯片已成为无细胞蛋白质合成的有用工具。在本研究中，我们尝试在微室阵列上利用体外蛋白质合成技术，从 DNA 文库中制造高度集成的蛋白质芯片，该微室阵列使用疏水性表面 PDMS（聚二甲基硅氧烷）和亲水性底部基板玻璃制成。这些结构特性防止了腔室之间的交叉污染。最小室的最小容积约为1pl。每个芯片的腔室总数在一个芯片上为 10,000 个（容量 150 pl），在另外两个芯片上（容量为 1 和 5 pl）为 250,000 个。接下来，我们尝试使用该微室阵列进行体外蛋白质合成。快速检测到室中表达的绿色荧光蛋白 (GFP) 的荧光（仅 1 小时内）。使用聚合物珠上固定的 DNA 分子也能成功表达 GFP。将 DNA 固定珠作为来源添加到每个微室中。蛋白质由 DNA 固定珠成功合成，这使得 DNA 分子的处理变得容易。
The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.