5'-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)- 3-acrylimido]-2'-deoxyUridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite
Deprotection: No changes needed from standard method recommended by synthesizer manufacturer.
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.
(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 spacer arm 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.