The LightCycler(TM) a microvolume multisample fluorimeter with rapid temperature control
The LightCycler(TM) a microvolume multisample fluorimeter with rapid temperature control
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DOI：
10.2144/97221pf02
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发表时间：
1997-01-01
期刊：
影响因子：
2.7
通讯作者：
Balis, UJ
中科院分区：
文献类型：
--
作者：
Wittwer, CT;Ririe, KM;Balis, UJ
本文介绍了实验用和商用的快速控温微体积荧光计。从流式细胞术中采用的荧光光学器件用于询问玻璃毛细管中的1-10 μ L样品。通过循环空气涡流获得均匀的温度控制和样品温度的快速变化（10摄氏度/秒）。一个原型2色，32样品的版本被构造与氙弧激发，单独的激发和发射路径，和光电倍增管检测。商业LightCycler（TM）是一种3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。应用包括分析物定量和核酸熔解曲线与荧光染料，酶测定与荧光底物和技术，使用荧光共振能量转移。微体积能力允许分析非常小或昂贵的样品。作为一种应用的实例，通过三种不同的荧光技术连续监测快速循环DNA扩增，所述荧光技术包括使用双链DNA染料绿色I、双标记的5 ′-外切核酸酶水解探针以及相邻的荧光素和Cy 5 TM标记的杂交探针。完整的扩增和分析仅需10-15分钟。
Experimental and commercial microvolume fluorimeters with rapid temperature control are described. Fluorescence optics adopted from flow cytometry were used to interrogate 1-10-mu L 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(TM), 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(R) Green I, a dual-labeled 5'-exonuclease hydrolysis probe, and adjacent fluorescein and Cy5(TM)-labeled hybridization probes. Complete amplification and analysis requires only 10-15 min.