Available modifications and turn around time
Roche Diagnostics suggests using only 5'-LightCycler® Red and 3'-fluorescein labeled probes. The synthesis or 3'-LightCycler® Red 640 probes and 5'-fluorescein probes is possible, but might lead to unsatisfactory results due to a different geometry influencing the FRET energy transfer.
For genotyping either the fluorescein or the LightCycler®-labeled probe can be used. The function of the 'sensor' is solely dependent on its lower melting temperature and its positioning spanning the variable site.
|
5'-LightCycler® Red 640 |
LC Red 640-aminohexyl-spacer-5'-OH. Synthesis of an amino modified product with 3'-phosphate, coupling with the dye, purification by HPLC and gel filtration. Spectrophotometric quantification at 260 nm. Production time three days. |
|
5'-LightCycler® Red 705 |
LightCycler® Red 705 is introduced by a phosphoramidite reagent (only 5'-label). Synthesis of an oligonucleotide product containing a 3'-phosphate, HPLC-purification and gel filtration. Spectrophotometric quantification. Production time three days. |
|
3'-Fluorescein (3FITC, 3FL) |
The fluorescein dye must contain a sulfur-linkage, as obtained from FITC-derived linkages. The color compensation of the LightCycler® is calibrated for this fluorescein derivate. Synthesis is carried out on a modified fluorescein-CPG. Purification by hydrophobic anion exchange chromatography and gel filtration. Spectrophotometric quantification.
Production time three days.
|
|
Internal LightCycler® Red 640 |
For applications requiring internal labels or amplification primers with an internal label (void of a 3'-block), the LC640 dye can be introduced at any internal Thymidin base (not available as 3'-modification). HPLC purification, spectrophotometric quantification. Production time three days. |
|
Quality control |
Hybridization probes developed or constructed by the design service of TIB MOLBIOL will be analyzed functionally using a melting curve routine on the LightCycler®. |
Arriba