Evolution of the LightCycler
The LightCycler began with a 1993 NIH STTR grant application to combine the optics of flow cytometry with rapid air thermal cycling. The glass capillaries of rapid cycling made ideal fluorescence cuvettes that were arranged in a carousel and rotated past stationary optics. The original prototype, affectionately named "Black Beauty", consumed 20 sq ft of bench space, and used a water cooled xenon arc source for excitation and photomultiplier tubes for detection. Temperature cycling was achieved with forced air using a hair dryer for heating and a household vacuum for cooling. In the first commercial version (LightCycler-24 by Idaho Technology) the bulky optical components were replaced by small LEDs and photodiodes for a final footprint of under 1 sq ft. The LightCycler was licensed to Roche in 1997, and was unveiled to the world in 1998. Over 20,000 LightCyclers have been sold, providing a rapid option for real-time PCR in 15-30 min. The first FDA approved kits for genetic variants (factor V Leiden and prothrombin) appeared on the LightCycler in 2003, and a 6-color version (the LC 2.0) in 2004. The product line was expanded in 2006 to include the LC480 system, a 96/384-well microtiter plate version that is not as fast, but allows higher throughput.
SYBR Green I | Real-Time PCR w/ Melting Analysis
The first LightCycler real-time curves used SYBR Green I to monitor target amplification. At that time (early in 1995), SYBR Green I was a dye just introduced by Molecular Probes for gel analysis of DNA. It had not been used for real-time PCR and was thought to be heat labile. However, its spectral properties (close to fluorescein) and high specificity to double-stranded DNA were much better than ethidium bromide. Contrary to the bias at the time, SYBR Green I worked very well for real-time PCR, and has become the most popular method of real-time monitoring for simple reasons of cost and convenience. Synthesis and labeling of probes became unnecessary. When SYBR Green I fluorescence is monitored once each cycle, the initial template concentration can be quantified. However, low concentrations of target can be difficult to quantify because unintended products may also be amplified and detected. These specificity concerns are not as important today because of hot-start methods and the common use of melting curve analysis to identify products.