Large-Scale Oligonucleotide Synthesis Using the Solid-Phase Approach

  • Protocol
Protocols for Oligonucleotides and Analogs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 20))

  • 3535 Accesses

Abstract

Synthetic DNA of defined sequences are commonly termed “oligonucleotides,” which are primarily composed of four different types of nucleosides linked through well-defined deoxyribose phosphate. Over the past ten years, because of refinement in synthesis chemistries (1a1e) and automation of synthesis steps (2a,2b), oligonucleotide syntheses of defined sequences are common practices in nonchemists’ laboratories. Oligonucleotides are routinely used as DNA sequencing primers (3a3c), probes (4a, 4b), linkers, adaptors, and gene synthesis (5). In addition to these applications, biophysical studies (6a,6b) (NMR, X-ray crystallography) for structural information of synthetic oligonucleotide using milligram quantities have been carried out. Newer applications of oligonucleotides are also emerging in the field of clinical diagnosis (7a7c), forensic testing, and disease treatment (8a8e). Investigations are being carried out in many laboratories for potential use of oligonucleotides as therapeutic agents broadly referred to as “use as antisense DNA.” Inhibition of viral replication in diseases, such as AIDS, herpes, and human pepiloma virus, and regulation of oncogene expression with oligonucleotides or their analogs are two major potential applications of synthetic DNA. Presently, these studies are moving out of research laboratories and into practical medical applications (9).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
EUR 44.95
Price includes VAT (Germany)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR 149.79
Price includes VAT (Germany)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
EUR 213.99
Price includes VAT (Germany)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info
Hardcover Book
EUR 192.59
Price includes VAT (Germany)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Beaucage, S.L. and Caruthers, M. H. (1981)Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859–1862.

    Article  CAS  Google Scholar 

  2. McBride, L. J. and Caruthers, M. H. (1983) Nucleotide chemistry, 10, an investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides. Tetrahedron Lett. 24, 245–248.

    Article  CAS  Google Scholar 

  3. Sinha, N. D., Biernat, J., and Köster, H. (1983) Beta-CYANOETHYL N,N-dialkylamino/N-Morpholinomonochloro phosphoramidites, new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides. Tetrahedron Lett. 24, 5843–5846.

    Article  CAS  Google Scholar 

  4. Sinha, N. D., Biernat, J., McManus, J. P., and Köster, H. (1984) Polymer support oligonucleotide synthesis, 18, use of beta-cyanoethyl-N,N-dialkyamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucl. Acids Res. 12, 4539–4557.

    Article  CAS  Google Scholar 

  5. Froehler, B. C., Ng, P. G., and Matteuccci, M. D. (1986) Synthesis of DNA via deoxynucleoside H-phosphonate intermediates. Nucl. Acids Res. 14, 5399–5407.

    Article  CAS  Google Scholar 

  6. Alvarado-Urbina, G., Sathe, G. M., Liu, W. C., Gillen, M. F., Duck, P. D., Bender, R., and Ogilvie, K. K. (1981) Automated synthesis of gene fragments. Science 214, 270–274.

    Article  CAS  Google Scholar 

  7. Hunkapillar, M., Kent, S., Caruthers, M., Dreyers, W., Firca, J., Griffin, C., Horvath, S., Hunkapillar, T., Tempest, P., and Hood, L. (1984) A microchemical facility for the analysis and synthesis of genes and proteins. Nature (Lond.) 310, 105–111.

    Article  Google Scholar 

  8. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980) Cloning in single stranded bacterio phage as an aid to rapid DNA sequencing. J. Mol. Biol. 143, 161–178.

    Article  CAS  Google Scholar 

  9. Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA sequencing. Nucl. Acids Res. 9, 309–321.

    Article  CAS  Google Scholar 

  10. Mullins, K. et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 51, 275.

    Google Scholar 

  11. Conner, B., Reyes, A., Morin, C., Itakura, K., Teplitz, R., and Wallace, R. (1983) Detection of sickle cell beta-S globin allele by hybridization with synthetic oligo nucleotides. Proc. Natl. Acad. Sci. USA 80, 278–282.

    Article  CAS  Google Scholar 

  12. Di, P., Meldon, P., Skingle, D. C., Lauser, J. A., and Symons, R. H. (1987) Enzyme-linked synthetic oligonucleotide probes—non-radioactive detection of entero-toxigenic escherichia-coli in fecal specimens. Nucl. Acids Res. 15, 5275–5287.

    Article  Google Scholar 

  13. Khorana, H. G. (1979) Total synthesis of a gene. Science 203, 614–625.

    Article  CAS  Google Scholar 

  14. Rich, A. et al. (1979) Molecular-structure of a left-handed double helical DNA fragment at atomic resolution. Nature 282, 680–686.

    Article  Google Scholar 

  15. Drew, A. R. and Dickerson, R. E. (1981) Structure of a B-DNA dodecamer, 3, geometry of hydration. J. Mol. Biol. 151, 535–556.

    Article  CAS  Google Scholar 

  16. Chehale, F. F., Doherty, M., Cai, S., Kan, Y. W., Cooper, S., and Rubin, E. M. (1987) Detection of sickle-cell anemia and thalassemias. Nature 329, 293,294.

    Google Scholar 

  17. Greenberg, S. J. Ehrlich, G. D., Abbott, M. A., Hurwitz, B. J., Waldmann, T. A., and Poiesz, B. J. (1989) Detection of sequences homologous to human retroviral DNA in multiple-sclerosis by gene amplification. Proc. Natl. Acad. Sci. USA 86, 2878–2882.

    Article  CAS  Google Scholar 

  18. Kogan, S. C., Doherty, M., and Gitschier, J. (1987) An improved method for prenatal-diagnosis of genetic-diseases by analysis of amplified DNA-sequences—application to hemophilia-A. N. Engl. J. Med. 317, 985–990.

    Article  CAS  Google Scholar 

  19. Stein, C. A. and Cohen, J. S. (1988) Oligodeoxynucleotides as inhibitors of gene-expression—a review. Cancer Res. 48, 2659–2668.

    CAS  Google Scholar 

  20. Heikkila, R., Schwale, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R., and Neckers, L. M. (1987) A C-MYC antisense oligodeoxynucleotide inhibits entry into S-phase but not progress from G0 to Gl. Nature 328, 445–449.

    Article  CAS  Google Scholar 

  21. Zamecnik, P. C., Goodchild, J., Taguchi, Y., and Sarim, P. C. (1986) Inhibition of replication and expression of human T cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA. Proc. Natl. Acad. Sci. USA 83, 4143–4146.

    Article  CAS  Google Scholar 

  22. Cooney, M., Czernuszewicz, G., Postel, E. H., Flint, S. J., and Hoga, M. E. (1988) Site-specific oligonucleotide binding represses transcription of the human C-MYC gene in-vitro. Science 241, 456–459.

    Article  CAS  Google Scholar 

  23. Toulene, J. J. and Helene, C. (1988) Antimessenger oligodeoxyribonucleo-tides—an alternative to antisense RNA for artificial regulation of gene-expression—a review. Gene 72, 51–58.

    Article  Google Scholar 

  24. Cohen, J. C. (1989) Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression. MacMillan, London.

    Google Scholar 

  25. Letsinger, R. L. and Mahadevan, V. (1965) Stepwise synthesis of oligodeoxyribonucleotides on an insoluble polymer support. J. Am. Chem. Soc. 87, 3526.

    Article  CAS  Google Scholar 

  26. Merrifield, R. B. (1965) Automated synthesis of peptides. Science 150, 178.

    Article  CAS  Google Scholar 

  27. Köster, H., Biernat, J., McManus, J., Wolter, A., Stumpe, A., Narang, C. K., and Sinha, N. D. (1984) Polymer support oligonucleotide synthesis, 15, synthesis of oligodeoxynucleotides on controlled pore glass (CPG) using phosphate and a new phosphite triester approach. Tetrahedron 40, 103–112.

    Article  Google Scholar 

  28. Wright, P., Lyttle, M., Carrol, J., Hudson, D., Warren, W., and Sinha, N. D., Large scale synthesis of oligodeoxynucleotides and polyphosphorothioated oligonucleotides, presented at the International Conference on Nucleic Acid Therapeutics held on January 13–17, 1991 in Clearwater Beach, Florida.

    Google Scholar 

  29. Letsinger, R. L. et al. (1988) Cationic oligonucleotides. J. Am. Chem. Soc. 110, 4470,4471.

    Article  CAS  Google Scholar 

  30. Iyer, R. P., Phillips, L. R., Eagon, W., Regan, J. B., and Beaucage, S. L. (1990) The automated synthesis of sulfur-containing oligodeoxyribonucleotides using 3H-1 2 benzodithiol-3-one 1 1-dioxide as a sulfur-transfer reagent. J. Org. Chem. 55, 4693–4699.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1993 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Sinha, N.D. (1993). Large-Scale Oligonucleotide Synthesis Using the Solid-Phase Approach. In: Agrawal, S. (eds) Protocols for Oligonucleotides and Analogs. Methods in Molecular Biology, vol 20. Humana Press. https://doi.org/10.1385/0-89603-281-7:437

Download citation

  • DOI: https://doi.org/10.1385/0-89603-281-7:437

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-281-1

  • Online ISBN: 978-1-59259-507-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Navigation