Glen Research biotin phosphoramidites for direct labelling of synthetic oligonucleotides exhibit the following features:
All are soluble in acetonitrile at concentrations useful for DNA synthesis.
All include a DMT group for cartridge puri cations which is essential for the preparation of biotinylated PCR primers because of the potential for cross contamination in HPLC puri cations.
For the development of diagnostic probes, biotin phosphoramidite is capable of branching to allow multiple biotins to be introduced at the 3’- or 5’-terminus. BiotinTEG Phosphoramidite contains a 15 atom mixed polarity spacer arm based on a triethylene glycol.
Coupling: 12-15 minute coupling time. To maintain label yield, carry out the synthesis DMT-on.
Deprotection: To maintain label yield as a 5' modifier, cleave and deprotect as required by nucleobases and then remove the DMT group. Alternatively, treat column with 10% diethylamine in acetonitrile for 2 minutes at room temperature (2x) to remove cyanoethyl protecting groups and rinse with ACN. At this point, the DMT group may be safely removed without loss of label during deprotection.
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.||
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.
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 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. ||