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.
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