Catalog Number: 10-1963-xx

Description: Fluorescein Phosphoramidite

FLUORESCEIN LABELLING

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.

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Material Safety Data Sheet

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(1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340.

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FREQUENTLY ASKED TECHNICAL QUESTION

QUESTION: Can oligonucleotides modified at the 5'-terminus with, for example, biotin be phosphorylated with kinase?

RESPONSE:Modification reagents based on a 1,2-diol, e.g., BioTEG, DNP phosphoramidites, or a 1,3-diol, e.g., fluorescein, biotin phosphoramidites, can be added several times at the 5'-terminus since they contain an alcohol group capable of further addition with phosphoramidites. Can this alcohol also be as a substrate for T4 polynucleotide kinase for 32P labelling of these modified oligonucleotides? Surprisingly, the answer is yes. Teoule and coworkers have shown(1) that oligos labelled at the 5'-terminus are substrates for kinase. Interestingly, the oligos modified with reagents based on 1,2-diols are labelled to 50%, indicating that only one diastereomer is labelled, while those modified with 1,3-diol reagents are labelled to 100%.

REFERENCE(S):
(1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340.

QUESTION: When quantifying a fluorescein labelled oligonucleotide by measuring absorbance at 260nm, what effect does the fluorescein have on this measurement?

RESPONSE:The extinction coefficient of DNA at 260nm is around 10,000/mole/base or 10/µmole/base. We use fluorescein isothiocyanate (Isomer 1) in the preparation of our fluorescein phosphoramidite (10-1963). The extinction coefficient of fluorescein (measured as FITC) at 260nm is around 13,700/mole or 13.7/µmole. The fluorescein contribution to the absorbance of a fluorescein labelled oligonucleotide at 260nm is, therefore, about the same as 1 base or about 5% of a 20mer. This may be neglected or corrected for in the determination of the amount of labelled oligonucleotide from an A260 measurement.

Note: dA=15.4, dC=7.4, dG=11.5, T=8.7

Fl-labeled ligos: 480nm excitation, 520nm emmission.

The extinction coefficient for fluorescein-5-isothiocyanate at 495 nm is 76,000 L/mole-cm at pH 9. Upon conjugation to protein, and by analogy oligo's, the extinction coefficient is decreased by 10%. This would result in a extinction coefficient of approximately 68,000 L/mole-cm. Additionally the extinction coefficient, and fluorescence emission, is very pH dependent (maximum at pH 9).

REFERENCE(S):
M. Powell, Glen Research

QUESTION: What are the relative extinction coefficients of 5'-Fluorescein, Hex and Tet etc.. at 260 nm and their Lambda max?

RESPONSE:Please see http://www.glenresearch.com/Technical/Extinctions.html

REFERENCE(S):
Oligonucleotide Properties Calculator; http://www.basic.northwestern.edu/biotools/oligocalc.html

QUESTION: Can oligonucleotides modified at the 5'-terminus with, for example, biotin be phosphorylated with kinase?

RESPONSE:Modification reagents based on a 1,2-diol, e.g., BioTEG, DNP phosphoramidites, or a 1,3-diol, e.g., fluorescein, biotin phosphoramidites, can be added several times at the 5'-terminus since they contain an alcohol group capable of further addition with phosphoramidites. Can this alcohol also be as a substrate for T4 polynucleotide kinase for 32P labelling of these modified oligonucleotides? Surprisingly, the answer is yes. Teoule and coworkers have shown(1) that oligos labelled at the 5'-terminus are substrates for kinase. Interestingly, the oligos modified with reagents based on 1,2-diols are labelled to 50%, indicating that only one diastereomer is labelled, while those modified with 1,3-diol reagents are labelled to 100%.

REFERENCE(S):
(1) M.L. Fontanel, H. Bazin, and R. Teoule, Analytical Biochemistry, 1993, 214, 338-340.

QUESTION: What are the relative extinction coefficients of various dyes?

RESPONSE:Please see http://www.glenres.com/Technical/Extinctions.html#dyes

QUESTION: Does AMA or methylamine cause any degradation to fluorescein or fluorescein-type dyes such as FAM or FITC?

RESPONSE: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%. The impurity is not detected with AMA at RT for 2 hours.

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DILUTION/COUPLING DATA

The table below shows pack size data and, for solutions, dilutions and approximate couplings based on normal priming procedures. Please link for more detailed usage information with the various synthesizers.

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200

If you cannot find the answer to your problem then please contact us or telephone +44 (0)1954 210 200