The LightCycler® Instrument (Figure) from Roche Diagnostics is an extremely fast thermocycler with on-line fluorescence detection. It enables the user to monitor the amplification of the PCR product simultaneously in real-time allowing the accurate quantification of target sequences. The innovative function called melting curve analysis performed after the PCR offers a powerful system for genotyping single nucleotide polymorphisms (SNPs) and for mutation detection.
1. The LightCycler® consists of a cylindrical chamber
where the air, which has virtually no mass, is warmed by a heating coil.
The PCR occurs in glass capillaries which can hold a volume of up to 20
µl. This high surface to volume ratio allows very rapid cycles.
2. The detection unit consists of a blue LED light source and three detection channels which measure emitted light at three different wave lengths (530 nm, 640 nm and 710 nm).
For more details on the LightCycler® Systems, including the newer generation LightCycler® 2.0 and the LightCycler® 480 please refer to www.roche-applied-science.com/lightcycler-online/.
Typical applications are the quantification of Interleukin transcripts in immunology, the detection of pathological mRNAs e.g bcrabl in CML Leukemia or the quantification of pathogenic germs.
Genotyping requires the use of hybridization probes. Examples of genotyping are the determination of genetic mutations as in Factor V Leiden responsible mutations, or genotyping of Herpes virus (HSV 1/2).
Several fluorescence formats are available to determine nucleic acids.