The Lightcycler

Carousel with capacity for 32 samples

Sealed 20 ul sample capillary with superior surface-to-volume ratio

At the beginning of amplification, the reaction mixture contains the denatured DNA, the

primers, and the dye.

After annealing of the primers, a few dye molecules can bind to the double strand. DNA binding results in a dramatic increase of the SYBR Green I molecules to emit light upon

excitation

During elongation, more and more dye molecules bind to the newly synthesized DNA

The amplification product can be detected by gel electrophoresis and ethidium bromide

staining

Essential components for using fluorescence-labeled oligonucleotides as Hybridization Probes

The first dye (fluorescein) is excited by the lightCycler’s LED ( light Emitting Diode) filtered light source , and emits green fluorescent light at

a slightly longer wavelength (middle figure)

This energy transfer , referred to as FRET (Fluorescence Resonance Energy Transfer ) is

highly dependent on the spacing between the two dye molecules

Using hybridization probes can also be beneficial if samples containing very few template molecules

are to be examined

Quantification with hybridization probes is not only sensitive but also highly specific

Single mismatch can significantly reduce the melting temperature of the oligonucleotide

Melting Curve Analysis: Hybridization probes matching the mutant and having a mismatch with the wild type.

Philadelphia translocation t(9;22) that is found is about 95% cases of chronic myelocytic leukemia (CML)

RT-PCR for CML using specific primers and hybridization probes for BCR-ABL translocation.