Matsubara Yasutaka, Kerman Kagan, Kobayashi Masaaki, Yamamura Shouhei, Morita Yasutaka, Takamura Yuzuru, Tamiya Eiichi
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa, 923-1292 Japan.
Anal Chem. 2004 Nov 1;76(21):6434-9. doi: 10.1021/ac0497149.
A novel method for multiplex TaqMan PCR in nanoliter volumes on a highly integrated silicon microchamber array is described. Three different gene targets, related to beta-actin, sex-determining region Y (SRY), and Rhesus D (RhD) were amplified and detected simultaneously on the same chip by using three different types of human genomic DNA as the templates. The lack of cross-contamination and carryover was shown using alternate dispensing of mineral oil-coated microchambers containing template and those without template. To confirm the specificity of our system to beta-actin, SRY, and RhD genes, we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The samples were cycled with the same sustaining temperatures as with the microchamber array. Instead of the conventional method of DNA quantification, counting the number of the fluorescence released microchambers in consequence to TaqMan PCR was employed to our chip. This simple method of observing the end point signal had provided a dynamic quantitative range. Stochastic amplification of 0.4 copies/reaction chamber was achieved. The microfabricated PCR chip demonstrated a rapid and highly sensitive response for simultaneous multiple-target detection, which is a promising step toward the development of a fully integrated device for the "lab-on-a-chip" DNA analysis.
本文描述了一种在高度集成的硅微腔阵列上进行纳升体积多重TaqMan PCR的新方法。使用三种不同类型的人类基因组DNA作为模板，在同一芯片上同时扩增和检测了与β-肌动蛋白、性别决定区Y（SRY）和恒河猴D（RhD）相关的三种不同基因靶点。通过交替分配含有模板的矿物油包被微腔和不含模板的微腔，证明了无交叉污染和残留。为了确认我们的系统对β-肌动蛋白、SRY和RhD基因的特异性，我们将较大体积的PCR样品用于商业实时PCR系统SmartCycler。样品在与微腔阵列相同的维持温度下进行循环。我们的芯片采用了一种不同于传统DNA定量方法的方式，即通过计数TaqMan PCR后释放荧光的微腔数量来进行定量。这种简单的观察终点信号的方法提供了一个动态定量范围。实现了每个反应腔0.4个拷贝的随机扩增。微制造的PCR芯片对同时进行多靶点检测表现出快速且高度灵敏的响应，这是朝着开发用于“芯片实验室”DNA分析的全集成设备迈出的有希望的一步。
