UNIT 4.20 Reversible Biotinylation of the 5′-Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification
Published Online: 1 NOV 2003
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Nucleic Acid Chemistry
How to Cite
Fang, S. and Bergstrom, D. E. 2003. Reversible Biotinylation of the 5′-Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification. Current Protocols in Nucleic Acid Chemistry. 14:4.20:4.20.1–4.20.17.
- Published Online: 1 NOV 2003
- Published Print: SEP 2003
The preparation of two reversible biotinylation phosphoramidites and their application in labeling and affinity purification of synthetic oligodeoxyribonucleotides will be described. In both cases, the biotin is linked to the 5′-terminus of DNA through a diisopropyl silyl acetal functionality. This linkage is completely stable under certain postsynthetic cleavage/deprotection conditions, but can be readily broken by fluoride ions, releasing unmodified 5′-OH and 5′-phosphate DNA, respectively. To demonstrate the use of these reversible biotinylation methods, crude DNA was incubated with NeutrAvidin-coated microspheres, full-length biotinylated DNA was efficiently attached to the solid phase, and nonbiotinylated failure sequences and other impurities were readily removed by washing with buffer. Cleavage of the silyl acetal linkage afforded high-quality, full-length, unmodified 5′-OH and 5′-phosphate DNA, respectively, depending on which of the two phosphoramidites was used. It is anticipated that this method will find applications in areas that require efficient isolation of DNA from a complex mixture.
- Fluoride Cleavage;
- Silyl Acetal;
- Affinity Purification