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5′‐Iodination of Solid‐Phase‐Linked Oligodeoxyribonucleotides
Publication Name:
Current Protocols in Nucleic Acid Chemistry
Unit Number:
Unit 4.19
DOI:
10.1002/0471142700.nc0419s14
Online Posting Date:
November, 2003 Abstract
5¢-Iodinated oligodeoxyribonucleotides readily react with 3¢-phosphorothioated DNA in the presence of a complementary template to yield a conjugate that is identical to natural DNA in every respect except that one oxygen atom in the phosphodiester backbone is replaced by a sulfur atom. The 5¢-iodo group is easily converted to a variety of other functional groups and will quickly react with thiol-containing labels to yield stable thioether conjugates. This unit presents manual and automated procedures for converting the 5¢-hydroxyl of protected CPG–bound oligodeoxyribonucleotides to an iodo group and for releasing and purifying the products.
Keywords: iodination; 5¢-modification; solid-phase; oligodeoxyribonucleotide; electrophile; ligation
Table of Contents
- Unit Introduction
- Basic Protocol: Manual Procedure for 5¢-Iodination of Oligodeoxyribonucleotides on a Solid Support
- Alternate Protocol: Automated Procedure for 5¢-Iodination of Oligodeoxyribonucleotides on a Solid Support
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol: Manual Procedure for 5¢-Iodination of Oligodeoxyribonucleotides on a Solid Support
Materials
- Anhydrous N,N-dimethylformamide (DMF)
- Iodination solution (see
- Methylene chloride (CH
2 Cl2 ) - 28% (v/v) ammonium hydroxide (NH
4 OH)
- DNA synthesizer (e.g., ABI; see appendix
- 0.2 to 10 µmol DNA synthesis column with long-chain alkylamine controlled-pore glass (CPG) support
- 1- and 10-mL syringes
- Shaker, rocker, or other agitating device
- C18 reversed-phase HPLC column (unit
- Speedvac evaporator
- Additional reagents and equipment for oligonucleotide synthesis (Chapters 3 and 4 and appendix
Alternate Protocol: Automated Procedure for 5¢-Iodination of Oligodeoxyribonucleotides on a Solid Support
Additional Materials (also see Basic Protocol )
- Glass wool or line filter (e.g., preparation and delivery line filter from ABI)
- Empty DNA synthesis column (e.g., 1000-Å CPG column; ABI)
- 10-mL syringes
- Clean, oven-dried phosphoramidite bottle compatible with synthesizer
- Reagent bottle compatible with synthesizer, filled with anhydrous DMF
Looking for materials? Suppliers Guide
Figures
-
Figure 4.19.1Iodination of solid-phase-linked oligodeoxyribonucleotides. Abbreviations: B1 and B2 , thymine and/or N-protected nucleobases; CPG, long-chain alkylamine controlled-pore glass (resin). -
Figure 4.19.2RP-HPLC profiles of 5¢-d(NGTAGGCAAGAGT) before and after 5¢-iodination. From top to bottom, N = T, C, A, and G, respectively. Reprinted from Miller and Kool (
Literature Cited
| Literature Cited | |
| Dimitrijevich, S.D., Verheyden, J.P.H., and Moffatt, J.G. 1979. Halo sugar nucleosides. 6. Synthesis of some 5¢-deoxy-5¢-iodo and 4¢,5¢-unsaturated purine nucleosides. J. Org. Chem. 44:400-406. | |
| Miller, G.P. and Kool, E.T. 2002. A simple method for electrophilic functionalization of DNA. Org. Lett. 4:3599-3601. | |
| Xu, Y. and Kool, E.T. 1997. A novel 5¢-iodonucleoside allows efficient non-enzymatic ligation of single-stranded and duplex DNAs. Tetrahedron Lett. 38:5595-5598. | |
| Xu, Y. and Kool, E.T. 1998. Chemical and enzymatic properties of bridging 5¢-S-phosphorothioester linkages in DNA. Nucl. Acids Res. 26:3159-3164. | |
| Key References | |
| Miller and Kool, 2002. See | |
| Reports a method to iodinate the 5¢-carbon of oligo-deoxyribonucleotides on a solid support. | |
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