5'-Fluorescein phosphoramidite contains no 4,4'-dimethoxytrityl (DMT) group and can be added only once at the 5'-terminus, thereby terminating synthesis. This product is prepared using the 6-carboxyfluorescein derivative. The tetrachloro (TET)-, hexachloro (HEX) - and dichloro-dimethoxy (JOE)- fluorescein phosphoramidites are designed to take advantage of the multicolor detection capability of modern DNA sequencers and genetic analyzers. Fluorescein phosphoramidite is designed to produce the same fluorescein-type structure as had been previously prepared using fluorescein isothiocyanate (FITC). Our fluorescein phosphoramidite also contains a DMT group to allow quantification of coupling. The analogous structure, 6-Fluorescein Phosphoramidite, prepared using 6-FAM, is also available, along with 6-Fluorescein Serinol Phosphoramidite. Fluorescein-dT can be inserted into the desired sequence as a replacement for a dT residue.
We offer five fluorescein supports. Fluorescein CPG has traditionally been used to add the fluorescein label at the 3'-terminus. The analogous structure, 3'-(6-Fluorescein) CPG, prepared using 6-FAM, is now also available, along with 6-Fluorescein Serinol CPG. We also offer 3'-(6-FAM) CPG and Fluorescein-dT CPG, both derivatives of 6-carboxyfluorescein (6-FAM). Both are single isomers and use an amide linkage which is stable during cleavage and deprotection and does not allow isomer formation. 3'-(6-FAM) CPG allows effective blockage of the 3'-terminus from polymerase extension as well as exonuclease digestion. Fluorescein-dT CPG allows both of these enzymatic activities to proceed. Normal cleavage and deprotection with ammonium hydroxide readily generates the fluorescein labelled oligos.
The spectral characteristics of these dyes are detailed on the following page.
Coupling: 15 minute coupling time recommended.
Deprotection: Use ammonium hydroxide and deprotect as required by nucleobases. When using AMA, a small amount of a non-fluorescent impurity will be formed. To eliminate this impurity, first deprotect with ammonium hydroxide for 30 minutes at room temperature, add an equal volume of 40% methylamine and then complete the deprotection as required by the nucleobases - e.g. 10 minutes at 65°C or 2 hours at room temperature for standard bases.
Response: While AMA (Ammonium hydroxide/40% Methylamine 1:1 v/v) is considered compatible with fluorescein, the use of methylamine when deprotecting a Fluorescein-labeled oligo does lead to a small amount of degradation, which is characterized by a the appearance of a late-eluting peak by RP HPLC that shows no visible fluorescein absorbance. With standard deprotection conditions (AMA 10 minutes at 65 C) the amount of degradation is approximately 5%|||