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Reporter groups on oligonucleotides |
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Fluorescently labeled oligonucleotides are used for:
IBA offers a broad spectrum of dyes for all of these applications.
There are different possibilities for labeling an oligonucleotide:
The delivery amount is 5 nmol* with the 0.05 µmol scale, 25* nmol with the 0.2 µmol* scale, and 100* nmol for the 1.0 µmol scale.
* for oligonucleotides shorter 40 bases
All dye labeled oligonucleotides are double HPLC purified (with the exception of the directly coupled dyes where no excessive dye must be eliminated). Failure sequences are removed during the first HPLC purification. The second HPLC is performed to remove unlabeled oligonucleotides and excessive dye. As a result only labeled oligonucleotides are obtained.
Labeling oligonucleotides with multiple dyes is also possible. This is used in Fluorescence Resonance Energy Transfer (FRET) applications.(1-3)
In theory, resonance energy transfer between two fluorophores will occur only if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. It was discovered, however, that "non- overlapping FRET pairs" can also be used, such as FAM/ Cy5TM or a wide range of fluorophores with Dabcyl as quencher. The fluorophores have to be able to reach close vicinity to each other. The excited fluorophore transfers the energy to the acceptor (quencher) without emission of a photon. For most assays fluorescein is used as a donor. Typical acceptor dyes are: Fluorescein, TAMRA, ROX, Cy5TM, Carboxy-rhodamine-6G, Dabcyl and QSY-7TM.
Reference
IBA offers a great variety of oligonucleotides with different labels and their combinations. For example: 5'-Fluorescein, 5'-HEX, 5'- Cy3TM, 5'- Cy3.5TM, 5'- Cy5TM, 5'- Cy5.5TM, 5'-TET, 3'-Dabcyl, and 3'-Fluorescein can be combined with any of the available dyes in varied positions. For double labelings with dyes that are not available as amidites labeling via thiol-reactive compounds is possible.
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