Reversible Biotinylation of the 5′‐Terminus of Oligodeoxyribonucleotides and its Application in Affinity Purification
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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
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- Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA
- Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA
- Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres
- Support Protocol 2: Synthesis of Biotinyl Alcohol
- Commentary
- Literature Cited
- Figures
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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐OH DNA
Materials
Basic Protocol 2: Reversible Biotinylation Via a Diisopropyl Silyl Acetal Linker Yielding 5′‐Phosphate DNA
Materials
Support Protocol 1: Application of the Reversible Biotinylation Method in Affinity Purification Using Avidin‐Coated Microspheres
Materials
Support Protocol 2: Synthesis of Biotinyl Alcohol
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Figure 4.20.1 Preparation of reversible biotinylation phosphoramidite S.3 and structures of biotinylated 5′‐OH DNAs S.4 and S.5 . View Image -
Figure 4.20.2 HPLC profiles of DNAs S.5 (trace a), 5′‐TACAGTGACT‐3′ (trace b), S.9 (trace c), and 5′‐H2 O3 PO‐ACAGTGACT‐3′ (trace d) generated on a C18 reversed‐phase column (100 Å, 250 × 4.6 mm, Varian Analytical Instruments), using a linear gradient of 0% to 45% solvent B (90% acetonitrile) in solvent A (0.1 M triethylammonium acetate, 5% acetonitrile) over 60 min at a flow rate of 1 mL/min by detecting the absorbance of DNA at 260 nm. For a detailed protocol for performing reversed‐phase HPLC, refer to UNIT . View Image -
Figure 4.20.3 Preparation of reversible biotinylation phosphoramidite S.7 and structures of biotinylated 5′‐phosphate DNAs S.8 and S.9 . View Image -
Figure 4.20.4 Removal of biotin from biotinylated DNAs S.4 and S.8 to generate unmodified 5′‐OH DNA S.10 and unmodified 5′‐phosphate DNA S.12 , respectively. View Image -
Figure 4.20.5 Preparation of the biotinyl alcohol S.1 . View Image
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Literature Cited
| Literature Cited | |
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