Single-molecule DNA amplification and analysis in an integrated microfluidic device
Single-molecule DNA amplification and analysis in an integrated microfluidic device
复制标题
DOI：
10.1021/ac001026b
复制
发表时间：
2001-02-01
影响因子：
7.4
通讯作者：
Mathies, RA
中科院分区：
文献类型：
--
作者：
Lagally, ET;Medintz, I;Mathies, RA
在集成微流体装置中演示了单个 DNA 模板分子的随机 PCR 扩增，然后对产物进行毛细管电泳 (CE) 分析。该微型装置由蚀刻在玻璃基板上的亚微升 PCR 室组成，直接连接到微型 CE 系统。阀门和疏水通风口为 280 nL PCR 室提供受控和无传感器加载；小容量反应器、低热质量和薄膜加热器的使用允许循环时间快至 30 秒。用嵌入荧光染料标记的扩增产物直接注入凝胶填充的毛细管通道中进行电泳分析。单 DNA 模板分子水平的重复 PCR 分析显示出量化的产物峰面积；归一化峰面积的直方图揭示了反应器中由 0、1、2 和 3 个可行模板副本引起的事件簇，并且这些事件簇显示符合泊松分布。该装置展示了微制造装置中最灵敏的 PCR。单个 DNA 分子的检测还将促进单细胞和单分子研究，以揭示整体序列和表达平均值背后的遗传变异。
Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s, The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.