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5'-Tetrachlorofluorescein phosphoramidite
Features and Biological Applications:
There are several ways of labeling an oligonucleotide with fluorescein. The choice of label is diversified further, depending on the spectral requirements. 5’-fluorescein-CE phosphoramidite, derived from the single isomer 6-carboxyfluorescein, 5’-hexachlorofluorescein-CE phosphoramidite (HEX) and 5’-tetrachlorofluorescein-CE phosphoramidite (TET) can all be used to efficiently label an oligonucleotide at the 5’-end. Labeling the 3’-end of an oligo with fluorescein can be achieved using 3’-Fluorescein CPGs. Standard cleavage and deprotection with ammonium hydroxide liberates the fluorescein-labeled oligo when using any of these supports.
6-TAMRA CPG *1000 Å*
The light-absorbing properties of TAMRA, and spectral overlap with several commonly used fluorophores - including FAM, HEX, TET and JOE, make it useful as a FRET acceptor for the dual labeled FRET probes such as Molecular Beacons. TAMRA has been used extensively for real-time PCR and other molecular diagnostic applications. Oligonucleotides can be labeled with TAMRA using two distinct methodologies. Under standard deprotection conditions, TAMRA is not sufficiently stable; the molecule degrades in the presence of ammonium hydroxide. If standard deprotection is required, the oligonucleotide is normally synthesized with an amino group at either the 3'-, or 5'-end and post-labeled with TAMRA succinimidyl ester. Oligonucleotides synthesized using UltraMILD monomers can also be labeled directly with TAMRA, either internally by substituting any suitable dT residue with TAMRA-dT-CE phosphoramidite, or at the 3'-end using 3'-TAMRA CPG support. Subsequent deprotection of the oligo using potassium carbonate in methanol adequately removes the base protecting groups, leaving the TAMRA intact. Alternatively the deprotection of 1:1:2 t-butylamine/methanol/water allows the use of regular monomers.
6-TET, SE
6-TET, SE is a popular amino-reactive fluorescent probe that is widely used in nucleic acid sequencing and related research.
6-HEX, SE
6-HEX, SE is the amine-reactive succinimidyl ester of 6-HEX acid. It is widely used in nucleic acid sequencing and related research.
Chemical Phosphorylation Reagent I (CPR I)
This reagent can be used to create the 3'-phosphate by adding it as the first addition to the support in Labeling Oligonucleotides with Fluorescent Dyes. After ammonia deprotection the oligo will have the 3'-phosphate attached to the 2nd base added during synthesis. Both the support base and the CPR are cleaved. The DMT-group is removed during the ammonium hydroxide deprotection and thus is not available for poly-pak purification.
Chemical Phosphorylation Reagent II (CPR II)
This Chemical Phosphorylation Reagent (CPR II) contains a DMT group that can be left on the oligonucleotide and used for rapid purification of oligonucleotide 5'-phosphates by the popular DMTr-on technique, which employs disposable RP cartridges or “Trityl-on” RP HPLC purification. The DMTr group is removed with aqueous acid (e.g., 2%TFA in the case of Cartridge Purification) and the remaining linker is then eliminated after brief treatment with aqueous ammonium hydroxide (12 -15% ammonium hydroxide at room temperature for 15 minutes) to yield the 5'-phosphate.
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