FABI Facilities: Roche LightCycler

Roche LightCycler
Figure 1. The LightCycler instrument. A total of 32 samples can be analysed at a time. The PCR reaction mixtures are loaded in disposable glass capillary tubes and arranged in a carousel (shown above) for thermo-cycling.

The Roche LightCycler was purchased in April 2002 by the Faculty of Natural and Agricultural Sciences, with substantial contributions by FABI and the Department of Genetics. The LightCycler is currently installed in room 6-21 of the Agricultural Sciences building and hosted by Dr. Zander Myburg.

Roche LightCycler
Figure 2. DNA quantification on the LightCycler system. A complete PCR of 25 to 30 cycles can be completed within 30 minutes, with DNA/dye quantity recorded after each cycle. The top panel shows the temperature profile, while the bottom panel shows increase in fluorescent signal over cycles in PCR reactions with a dilution series of the same DNA template.

The most important application of the LightCycler is to quantify DNA and RNA by real-time, quantitative PCR. The system allows a wide range of detection formats such as the DNA binding dye SYBR Green I, or fluorescently labelled hybridization probes. The LightCycler system monitors the progress of DNA amplification at each cycle of the PCR reaction (see Figure 2) and uses the number of PCR cycles required to amplify the PCR product above background to estimate the quantity of starting template (relative to a standard or reference template).

Roche LightCycler
Figure 3. Mutation detection by melting curve analysis. Top panel: melting curves of amplified gene fragments from wild-type and mutant individuals. Bottom panel: negative first derivatives of the melting curves showing the unique melting peak of each genotype. The gene fragment amplified from the heterozygous individual exhibits a melting curve with both the wild-type and mutant peaks (red plot).

The LightCycler also allows mutation detection by melting curve analysis (see Figure 3). This analysis allows discrimination of different amplification products based on the unique melting temperature of templates with even single nucleotide differences. In diploid organisms, heterozygotes can be distinguished from homozygotes.

For more information visit the LightCycler Homepage at:
http://www.roche-applied-science.com/lightcycler-online/

All images on this page are © 2000-2002 Roche Diagnostics Corporation.

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