Unit

UNIT 4.20 Reversible Biotinylation of the 5′-Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification

  1. Shiyue Fang1,2,
  2. Donald E. Bergstrom1,2

Published Online: 1 NOV 2003

DOI: 10.1002/0471142700.nc0420s14

Current Protocols in Nucleic Acid Chemistry

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.

Author Information

  1. 1

    Purdue University, West Lafayette, Indiana

  2. 2

    Walther Cancer Institute, Indianapolis, Indiana

Publication History

  1. Published Online: 1 NOV 2003
  2. Published Print: SEP 2003

Abstract

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.

Keywords:

  • DNA;
  • Phosphoramidite;
  • Biotinylation;
  • Fluoride Cleavage;
  • Silyl Acetal;
  • Avidin;
  • Affinity Purification