The LightCycler ^TM : A Microvolume Multisample Fluorimeter with Rapid Temperature Control

TLDR

Applications include analyte quantification, nucleic acid melting curves with fluorescent dyes, enzyme assays with fluorescent substrates, and fluorescence resonance energy transfer techniques. The study describes experimental and commercial microvolume fluorimeters with rapid temperature control. The instruments use flow‑cytometry‑derived fluorescence optics to interrogate 1–10 µL samples in glass capillaries, achieve homogeneous temperature control with a circulating air vortex at 10 °C s⁻¹, and employ either a xenon arc or blue LED excitation with separate excitation/emission paths and photomultiplier tubes or photodiodes for detection; a prototype 2‑color 32‑sample version and a commercial 3‑color 24‑sample LightCycler were built, and rapid cycle DNA amplification was monitored by SYBR Green I, a dual‑labeled 5′‑exonuclease probe, and adjacent fluorescein/Cy5z‑labeled hybridization probes. The microvolume capability permits analysis of very small or expensive samples, enabling complete DNA amplification and analysis in only 10–15 min.

Abstract

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.