The Lightcycler
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Transcript of The Lightcycler
![Page 1: The Lightcycler](https://reader034.fdocuments.us/reader034/viewer/2022050805/56812b88550346895d8fa664/html5/thumbnails/1.jpg)
The Lightcycler
![Page 2: The Lightcycler](https://reader034.fdocuments.us/reader034/viewer/2022050805/56812b88550346895d8fa664/html5/thumbnails/2.jpg)
Carousel with capacity for 32 samples
![Page 3: The Lightcycler](https://reader034.fdocuments.us/reader034/viewer/2022050805/56812b88550346895d8fa664/html5/thumbnails/3.jpg)
Sealed 20 ul sample capillary with superior surface-to-volume ratio
![Page 4: The Lightcycler](https://reader034.fdocuments.us/reader034/viewer/2022050805/56812b88550346895d8fa664/html5/thumbnails/4.jpg)
At the beginning of amplification, the reaction mixture contains the denatured DNA, the
primers, and the dye.
![Page 5: The Lightcycler](https://reader034.fdocuments.us/reader034/viewer/2022050805/56812b88550346895d8fa664/html5/thumbnails/5.jpg)
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
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During elongation, more and more dye molecules bind to the newly synthesized DNA
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The amplification product can be detected by gel electrophoresis and ethidium bromide
staining
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Essential components for using fluorescence-labeled oligonucleotides as Hybridization Probes
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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)
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This energy transfer , referred to as FRET (Fluorescence Resonance Energy Transfer ) is
highly dependent on the spacing between the two dye molecules
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Using hybridization probes can also be beneficial if samples containing very few template molecules
are to be examined
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Quantification with hybridization probes is not only sensitive but also highly specific
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Single mismatch can significantly reduce the melting temperature of the oligonucleotide
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Melting Curve Analysis: Hybridization probes matching the mutant and having a mismatch with the wild type.
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Philadelphia translocation t(9;22) that is found is about 95% cases of chronic myelocytic leukemia (CML)
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RT-PCR for CML using specific primers and hybridization probes for BCR-ABL translocation.