Oligo Synthesis : CEPs

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 seehttp://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 seehttp://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 formore detailed usage informationwith the various synthesizers.

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