Key featuresShow Hide
- Used to add a green-blue fluorescent dye to the 3' end of an oligonucleotide.
- Maximal emission around 525 nm.
- Quenched by BHQ-1, DDQ-1 and dabcyl.
- Derived from the single isomer 6-carboxyfluorescein.
- Does not block the 3'-terminus from polymerase extension, or prevent exonuclease activity.
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Fluorescein dyes fluoresce in the green-blue region of the visible spectrum, and are most efficiently quenched by BHQ-1. There are several ways of labelling an oligonucleotide with fluorescein-type dyes, and the choice of label is diverse, depending on the degree of chlorination of the aromatic rings. This determines the fluorescence emission of the dye.
5'-Fluorescein-CE Phosphoramidite (or “6-FAM”), derived from the single isomer 6 carboxyfluorescein is probably the most commonly used phosphoramidite, but 5'-Hexachloro-fluorescein-CE Phosphoramidite (HEX) and 5’ Tetrachlorofluorescein-CE Phosphoramidite (TET), can also be used to efficiently label an oligonucleotide at the 5'-end, with differing emission maxima.
While both 6-Fluorescein-CE Phosphoramidite and Fluorescein-CE Phosphoramidite incorporate the same fluorescent dye as 6-FAM, the linking backbone differs. The former has a 1,3-diol structure, where the additional OH is protected with DMTr. This not only allows coupling efficiency monitoring by DMTr release, it allows the possibility of multiple additions within the oligo for use in, e.g. chromosome painting. However, this often requires a linker (e.g. spacer-18) to be incorporated between each addition to prevent self-quenching of fluorescein. In the same way spacer-C3 is used to mimic the distance between the 3’ and 5'-O of dR, the 1,3-diol arrangement of 6-Fluorescein-CE Phosphoramidite provides the same scenario. It must be noted that, as with spacer-C3, a distortion of the backbone occurs, particularly with multiple incorporations.
Fluorescein-CE Phosphoramidite has a thiourea linkage which mimics the original method of incorporating fluorescein to an amino-modified oligo. It must be noted however that the linkage is attached via the 5 position of the ring system in this case.
Internal sequence additions of Fluorescein are achieved using Fluorescein-dT. Again, multiple additions can be carried out but the spacing between each fluorescein-dT is crucial to prevent self-quenching.
Labelling the 3'-end of an oligo with fluorescein can be achieved by a variety of solid supports, with spacer and dT options. Notably, the 3'-(6-FAM) CPG also allows the effective blockage of the 3'-terminus from polymerase extension, as well as exonuclease activity.
Lastly, 5,6-Fluorescein-OH carboxylic acid product is a mixed isomer free acid which can be used for labelling free amines on proteins.
|LK2134||5'-Fluorescein-CE Phosphoramidite (6-FAM)|
|LK2136||5'-Hexachloro-Fluorescein-CE Phosphoramidite (HEX)|
|LK2137||5'-Tetrachloro-Fluorescein-CE Phosphoramidite (TET)|
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050). Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
6-FAM (LK2134), HEX (LK2136) & TET (LK2137) – A 3min coupling time is recommended.
LK2148 – A 12-15min coupling time is recommended.
LK2139 – A 15min coupling time is recommended.
LK2068 – A 10min coupling time is recommended.
All CPG supports are used in a manner identical to standard nucleoside supports.
Non-nucleosidic CPG supports do not detritylate as rapidly as nucleosidic ones, therefore an additional detritylation step is recommended. It is therefore necessary to use a cycle that does not contain an initial capping step.
LK2134, LK2137, LK2148 & LK2139 can be deprotected following standard protocols with ammonium hydroxide solution, although LK2137 should not be subjected to prolonged heating at 55°C.
LK2136 is also deprotected with ammonium hydroxide solution, but at RT for 24h. The ammonia must be removed immediately after deprotection. Do not use tbutylamine/methanol/water (1:1:2) as this will completely degrade the HEX.
Oligos containing LK2068 can be deprotected with ammonium hydroxide using standard conditions, removing the fluorescein protecting groups at the same time.
LK2366, LK2359 & LK2368 – Cleavage of the oligonucleotide from these supports requires 45min at room temperature with ammonium hydroxide. Complete the deprotection using the protocol required by the nucleobases.
LK2370 – Deprotect using protocols required by the nucleobases.
Fluorescently labelled oligonucleotides can be purified and analysed using the same methods employed for standard DNA. Note, however, DMT ON purifications are not possible with LK2134, LK2136 or LK2137 as these are 5’-modifications with no DMTr group. Nevertheless, the hydrophobic nature of these modifications make purification straightforward.
It is also possible to carry out analysis on dilute solutions using fluorescence detection with RP-HPLC (see below for data).
Storage & Stability
All phosphoramidites and CPGs are stored dry, frozen at –10°C to –30°C. 2134 and 2137 are stable in solution for 1-2 days and after 4 days show <90% coupling efficiency. LK2139 is stable in solution for 2-3 days, LK2068 & LK2136 only for 24h, and LK2148 is unstable and must be used immediately after preparation.
Fluorescently labelled oligonucleotides must be stored in the dark, either dry or in neutral aqueous media at –20°C. Do not store crude fluorescently labelled oligonucleotides in ammonium hydroxide solution.
Fluorescein Labelling of RNA
It is possible to label an RNA oligo at the 3’- or 5’-end with fluorescein and deprotect/desilylate leaving the label intact, provided the desilylation is carried out using Et3N.3HF in DMSO (1:1) in place of TBAF.
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