Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber
Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber
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DOI：
10.1007/s10544-008-9163-x
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发表时间：
2008-02
影响因子：
2.8
通讯作者：
Liza Lam;S. Sakakihara;K. Ishizuka;S. Takeuchi;H. Arata;H. Fujita;H. Noji
中科院分区：
文献类型：
--
作者：
Liza Lam;S. Sakakihara;K. Ishizuka;S. Takeuchi;H. Arata;H. Fujita;H. Noji
环介导等温扩增（LAMP）是Notomi等建立的一种新颖的核酸扩增方法，在等温条件下进行，仅采用基本的反应流程和最少的辅助电子设备。这些要求被证明是可行的探索途径，以缩小规模的这种生物反应的实验室芯片上的应用。因此，在这里，我们开发了一种新的技术，在单分子水平的LAMP荧光成像。实验在聚丙烯酰胺（PAA）凝胶基微室中进行，其中最初包封自由悬浮于含有引物和聚合酶的溶液中的单个DNA模板。为了激活扩增反应，使用由自动计算机化反馈系统调节的微加热器进行局部加热。这种基于微室的LAMP方法证明了微量模板和引物的有效利用，以及LAMP检测时间的总体减少。在65°C下孵育50分钟后进行DNA扩增的平均效率为80%。由于包括检测在内的反应总时间可以在不到1小时内完成，这种一步直接观察方法显示出作为临床实验室遗传分析和诊断常规技术的简单替代方法的潜力。
Loop-mediated isothermal amplification (LAMP) is an original nucleic acid amplification method established by Notomi et al. LAMP is performed under isothermal condition, employing only a basic reaction protocol and minimal supporting electronics. These requirements prove to be viable for exploring the avenues to down-scale this biological reaction for Lab-on-a-chip application. Hence here, we developed a novel technique for fluorescent imaging of LAMP at a single molecule level. The experiment was conducted in a polyacrylamide (PAA) gel-based microchamber where a single DNA template, freely suspended in a solution containing primers and polymerase was initially encapsulated. In order to activate the amplification reaction, a microheater regulated by an automatic computerized feedback system was used for localized heating. This microchamber-based approach for LAMP demonstrated the effective exploitation of minute amount of templates and primers, and the overall reduction in LAMP detection time. An average efficiency of 80% was evaluated for conducting DNA amplification after 50 min of incubation at 65°C. As the total time for reaction including detection can be completed in less than 1 h, this one-step, direct observation method displays the potential as a simple alternative to conventional techniques for genetic analysis and diagnosis in the clinical laboratory.