Available modifications and turn around time

Roche Diagnostics suggests using only 5'-LightCycler® Red and 3'-fluorescein labeled probes. The synthesis of 3'-LightCycler® Red 640 probes and 5'-fluorescein probes is possible, but can 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 derivative. Synthesis is carried out on a modified fluorescein-CPG. Purification by hydrophobic anion exchange chromatography and gel filtration. Spectrophotometric quantification.

Production time three days.

farbstoff

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® Instrument.