|SIMA (HEX) Phosphoramidite
Catalog Number: 10-5905-xx
Description: SIMA (HEX) Phosphoramidite
Diluent: Anhydrous Acetonitrile
|Coupling: 3 minute coupling time recommended.
|Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.
|Storage: Freezer storage, -10 to -30°C, dry
|Stability in Solution: 1-2 days, <90% efficient after 4 days
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-, hexachloro-and dichloro-dimethoxy-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.
FLUORESCEIN LAELLING (SIMA)
Dichloro-diphenyl-fluorescein, SIMA (HEX) exhibits virtually identical absorbance and emission spectra to HEX. SIMA (HEX) is much more stable to basic deprotection conditions than HEX and oligonucleotides can be deprotected using ammonium hydroxide at elevated temperatures and even ammonium hydroxide/methylamine (AMA) at room temperature or 65°C for 10 minutes. SIMA absorption maximum was 3 nm blue-shifted compared to HEX at pH 7. The absorbance is broader, so the extinction coefficient is smaller than that of HEX, but when exciting at 500 nm where the absorbance was normalized, the emission was still 90% of HEX and the emission was red-shifted by 5 nm. A second SIMA (HEX) product, SIMA (HEX)-dT, can be used to introduce SIMA (HEX) in the synthetic oligonucleotide sequence, usually as a replacement for the native dT linkage. Again, this product is fully compatible with deprotection schemes using ammonium hydroxide at elevated temperatures or AMA at room temperature and 65°C.
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