Wittwer C T, Ririe K M, Andrew R V, David D A, Gundry R A, Balis U J
Idaho Technology, Idaho Falls, USA.
Biotechniques. 1997 Jan;22(1):176-81. doi: 10.2144/97221pf02.
Experimental and commercial microvolume fluorimeters with rapid temperature control are described. Fluorescence optics adopted from flow cytometry were used to interrogate 1-10-microL samples in glass capillaries. Homogeneous temperature control and rapid change of sample temperatures (10 degrees C/s) were obtained by a circulating air vortex. A prototype 2-color, 32-sample version was constructed with a xenon arc for excitation, separate excitation and emission paths, and photomultiplier tubes for detection. The commercial LightCycler, a 3-color, 24-sample instrument, uses a blue light-emitting diode for excitation, paraxial epi-illumination through the capillary tip and photodiodes for detection. Applications include analyte quantification and nucleic acid melting curves with fluorescent dyes, enzyme assays with fluorescent substrates and techniques that use fluorescence resonance energy transfer. Microvolume capability allows analysis of very small or expensive samples. As an example of one application, rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques, Which included using the double-stranded DNA dye SYBR Green I, a dual-labeled 5'-exonuclease hydrolysis probe, and adjacent fluorescein and Cy5z-labeled hybridization probes. Complete amplification and analysis requires only 10-15 min.
本文描述了具有快速温度控制功能的实验型和商用微量荧光计。采用流式细胞术的荧光光学元件对玻璃毛细管中的1 - 10微升样品进行检测。通过循环空气涡旋实现均匀温度控制和样品温度的快速变化（10℃/秒）。构建了一个原型双色32样品版本，采用氙弧灯进行激发，具有独立的激发和发射路径，以及用于检测的光电倍增管。商用的LightCycler是一种三色24样品仪器，使用蓝色发光二极管进行激发，通过毛细管尖端进行近轴落射照明，并使用光电二极管进行检测。其应用包括使用荧光染料进行分析物定量和核酸熔解曲线分析、使用荧光底物进行酶分析以及采用荧光共振能量转移的技术。微量检测能力允许对非常小的或昂贵的样品进行分析。作为一个应用实例，通过三种不同的荧光技术对快速循环DNA扩增进行连续监测，这三种技术包括使用双链DNA染料SYBR Green I、双标记的5' - 外切核酸酶水解探针以及相邻的荧光素和Cy5标记的杂交探针。完整的扩增和分析仅需10 - 15分钟。
