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: 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.

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.

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.

11/04/2011 | http://www.glenres.com/ProductFiles/10-5950.html