The Lightcycler

1. The Lightcycler

2. Carousel with capacity for 32 samples

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

4. At the beginning of amplification, the reaction mixture contains the denatured DNA, the primers, and the dye.

5. 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

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

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

8. Essential components for using fluorescence-labeled oligonucleotides as Hybridization Probes

9. 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)

10. This energy transfer , referred to as FRET (Fluorescence Resonance Energy Transfer ) is highly dependent on the spacing between the two dye molecules

11. Using hybridization probes can also be beneficial if samples containing very few template molecules are to be examined

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

13. Single mismatch can significantly reduce the melting temperature of the oligonucleotide

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

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

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