Oligo Synthesis : CEPs

Catalog Number: 10-1038-xx

Description: Biotin-dT

Biotin-dT can replace dT residues within the oligonucleotidesequence. 5’-Biotin phosphoramidite can be added ONLY ONCE to the 5’-terminus ofan oligonucleotide. However, the DMT group on the biotin can be used in RPcartridge and HPLC purification techniques. PC Biotin is a photocleavable5’-biotin phosphoramidite. BiotinTEG CPG and Protected BiotinLC Serinol CPG aredesigned for the direct synthesis of oligonucleotides containing biotin at the3’ terminus.

Desthiobiotin is a biotin analogue that exhibits lower binding tobiotin-binding proteins such as streptavidin. This biotin analogue is lackingthe sulfur group from the molecule and has a dissociation constant (Kd) severalorders of magnitude less than biotin/streptavidin. As a result, biomoleculescontaining desthiobiotin are dissociated from streptavidin simply in thepresence of buffered solutions of biotin. We offer desthiobiotinTEGphosphoramidite and the corresponding CPG.

ABI-style vials and columns are supplied unless otherwise requested.

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Material Safety Data Sheet

Glen Report 6.1: DEOXYURIDINE DERIVATIVES FOR INTERNAL MODIFICATION

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Frequently Asked Technical Question

QUESTION: 1.If Biotin-dT is inserted at the 5"-terminus, will it affect the function of kinase?2.Does Biotin-dT affect hybridization?

RESPONSE:1.Biotin dT will not affect the ability of kinase to phosphorylate at the 5"-terminus.

2.The presence of Biotin-dT within an oligonucleotide does not affect hybridization relative to the same oligo containing dT(1).The biotin resides in the major groove of the formed double-stranded DNA and is readily available for binding to avidin or streptavidin.

REFERENCE(S): (1)J. Telser,K.A. Cruickshank,L.E. Morrison, and T.L. Netzel, J. Am. Chem. Soc., 1989, 111, 6966-6976.

QUESTION: What absorbance does biotin have at 260nm?The HPLC trace shows absorbance at 254nm?

RESPONSE:Biotin is transparent at 260nm.The UV detector in the HPLC trace of biotin phosphoramidites is monitoring the absorption of the DMT group on the spacer arm.

QUESTION: Do you have a biotin phosphoramidite containing a disulfide linker which can be cleaved later with DTT to release the DNA from a streptavidin support?

RESPONSE:No.However, this can be produced on the synthesizer by adding to the 5"- terminus first 5"-thiol-modifier C6 S-S (10-1936) followed by BioTEG phosphoramidite (10-1955).This should generate a biotinylated primer with a long spacerarm containing the disulfide linkage which can be cleaved later with DTT.

QUESTION: How can I tell if the biotinylated oligonucleotide I have made really does contain biotin?

RESPONSE:A colorimetric assay for biotin can be quite effective.The color results from the reaction of biotin with p-dimethylaminocinnamaldehyde in the presence of sulfuric acid.

1.Spot 0.2 A260 units of biotinylated oligonucleotide on a silica gel TLC plate.

2.Dry the plate.]

3.Spray with a solution of 2% p-dimethylaminocinnamaldehyde (Sigma), 2% conc. sulfuric acid in ethanol.

4.Heat the plate and the presence of biotin will be indicated by the formation of a pink spot.

Since the intensity of the biotin spot is quite low, it is prudent to compare with an unlabelled oligonucleotide similarly treated.

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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.

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