Biotin oligonucleotide labeling reactions: A method to assess their effectiveness and reproducibility
| Autor: | Erika Reitano, Aldo Di Vito, Margherita Iaboni, Luisa Poggi |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Streptavidin
Oligonucleotides Biophysics Biotin 01 natural sciences Biochemistry 03 medical and health sciences chemistry.chemical_compound Protein purification Biotinylation Molecular Biology 030304 developmental biology chemistry.chemical_classification Detection limit 0303 health sciences Staining and Labeling Oligonucleotide Biomolecule 010401 analytical chemistry Cell Biology Combinatorial chemistry 0104 chemical sciences Kinetics chemistry Nucleic acid RNA |
| Zdroj: | Analytical Biochemistry. 593:113590 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2020.113590 |
| Popis: | The strong molecular interaction between biotin and streptavidin is widely used in the growing field of nucleic acid nanotechnology. Several biotin labeled oligonucleotide tools have been developed for the detection of biological molecules as well as for protein purification. For these reasons, biotinylation can be considered one of the main chemical reactions for nucleic acid labeling. However, despite its widespread application and the presence on the market of many reagents for the conjugation of biotin to oligonucleotides, it is not yet available a cheap, easy and sensitive system able to assess the effectiveness and reproducibility of this reaction. Here, we present an accurate and reliable method to achieve a qualitative and quantitative analysis of oligonucleotide biotinylation. The protocol employs basic laboratory instruments and standard software for molecular biology applications and does not require advanced expertise for its execution. Most importantly, our method is independent from complex kinetic equilibrium parameters and shows a limit of detection more than one order of magnitude lower than the current fluorometric gold standard assay. Therefore, this method could become a standard, inexpensive and routinely used quality test for post-synthesis evaluation of biotin conjugation reactions. |
| Databáze: | OpenAIRE |
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