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 (1a–1e) 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 (3a–3c), 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 (7a–7c), forensic testing, and disease treatment (8a–8e). 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).
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References
Beaucage, S.L. and Caruthers, M. H. (1981)Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859–1862.
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.
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.
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.
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.
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.
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.
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.
Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA sequencing. Nucl. Acids Res. 9, 309–321.
Mullins, K. et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 51, 275.
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.
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.
Khorana, H. G. (1979) Total synthesis of a gene. Science 203, 614–625.
Rich, A. et al. (1979) Molecular-structure of a left-handed double helical DNA fragment at atomic resolution. Nature 282, 680–686.
Drew, A. R. and Dickerson, R. E. (1981) Structure of a B-DNA dodecamer, 3, geometry of hydration. J. Mol. Biol. 151, 535–556.
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.
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.
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.
Stein, C. A. and Cohen, J. S. (1988) Oligodeoxynucleotides as inhibitors of gene-expression—a review. Cancer Res. 48, 2659–2668.
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.
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.
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.
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.
Cohen, J. C. (1989) Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression. MacMillan, London.
Letsinger, R. L. and Mahadevan, V. (1965) Stepwise synthesis of oligodeoxyribonucleotides on an insoluble polymer support. J. Am. Chem. Soc. 87, 3526.
Merrifield, R. B. (1965) Automated synthesis of peptides. Science 150, 178.
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.
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.
Letsinger, R. L. et al. (1988) Cationic oligonucleotides. J. Am. Chem. Soc. 110, 4470,4471.
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.
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© 1993 Humana Press Inc., Totowa, NJ
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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
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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
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