Manual Oligonucleotide Synthesis Using the Phosphoramidite Method

  • H. A. White
Part of the Methods in Molecular Biology book series (MIMB, volume 4)

Abstract

Oligodeoxyribonucleotides may be synthesized on a solid support, which allows for the elongation of the chain without intermediate purifications. The exocyclic amino groups on cytosine and the purines are protected as the alkali-labile benzoyl or iso-butyryl amides. The 3′-OH group of the first nucleoside is attached to the solid support by a spacer chain, and the synthesis procedes by coupling the 5′-OH group of the growing chain to the 3′-phosphorus of the monomer being added, which has its 5′-OH group protected by an acid-labile group. The phosphorus must also be protected to avoid side reactions. When the synthesis is completed, the chain must be cleaved from the support and the base, phosphorus, and terminal 5′-OH-protecting groups removed. The desired oligonucleotide is then separated from the mixture of shorter chains and modified chains (1).

References

  1. 1.
    Gait, M. J. (1984) An Introduction to Modern Methods of DNA Synthesis, in Oligonucleotide Synthesis—A Practical Approach (Gait, M. J., ed.) IRL, Oxford and Washington.Google Scholar
  2. 2.
    Atkinson, T. and Smith, M. (1984) Solid-Phase Synthesis of Oligodeoxyribonucleotidesby the Phosphite-Triester Method, in Oligonucleotide Synthesis—A practical Approach (Gait, M. J., ed.) IRL, Oxford.Google Scholar
  3. 3.
    Sinha, N. D., Biernat, J., McManus, J., and Koster, H. (1984) Polymer support oligonucleotide synthesis. XVIII. Use of (β-cyanoethyl-N,N-dialkylairrino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 12, 4539–4557.PubMedCrossRefGoogle Scholar
  4. 4.
    Jones, R. A. (1984) Preparation of Protected Deoxyribonucleosides, in Oligonucleotice Synthesis—A Practical Approach (Gait, M. J. ed.) IRL, Oxford.Google Scholar
  5. 5.
    Cruachem Ltd. (1986) Cyanoethylphosphoramidites. Cruachem Highlights, May 1986.Google Scholar
  6. 6.
    Jiricny, J. and Jones, M. B. (1984) The use of complementary 5′ protecting groups, in the synthesis of mixed oligodeoxyribonucleotide sequences.Google Scholar
  7. 7.
    Gait, M. J., Matthes, H. W. D., Singh, M., Sproat, B. S., and Titmas, R. C. (1982) Synthesis of Oligodeoxyribonucleotides by a Continuous Flow, Phosphotriester Method on a Kieselguhr/Polyamide Support, in Chemical and Enzymatic Synthesis of Gene Fragments (Gassen, H. G. and Lang, A., eds.) Verlag Chemie, Basel.Google Scholar
  8. 8.
    Gaastra, W. and Josephsen, J. (1984) Radiolabeling of DNA Using Polynucleotide Kinase, in Methods in Molecular Biology Vol. 2. Nucleic Acids (Walker, J. M., ed.) Humana, Clifton.Google Scholar
  9. 9.
    McLaughlin, L. W. and Piel, N. (1984) Chromatographic Purification of Synthetic Oligonucleotides, in Oligonucleotide Synthesis — A Practical Approach (Gait, M. J., ed.) IRL, Oxford.Google Scholar

Copyright information

© The Humana Press Inc. 1988

Authors and Affiliations

  • H. A. White
    • 1
  1. 1.Department of BiochemistryUniversity College LondonLondonUK

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