Synthesis and Purification of Oligonucleotides

Andrew Ellington1, Jack D. Pollard2

1 University of Texas, Austin, Texas, 2 Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.11
DOI:  10.1002/0471142727.mb0211s42
Online Posting Date:  May, 2001
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This unit provides strategies on the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection of DNA and RNA oligonucleotides.

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Table of Contents

  • Introduction to Chemical Nucleic Acid Synthesis
  • Strategies for Nucleic Acid Synthesis
  • Strategies for Oligonucleotide Purification
  • Basic Protocol 1: Monitoring DNA Synthesis Using the Trityl Assay
  • Support Protocol 1: Using the Trityl Assay for Troubleshooting
  • Basic Protocol 2: Deprotection of DNA Oligonucleotides
  • Basic Protocol 3: Deprotection of RNA Oligonucleotides
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Monitoring DNA Synthesis Using the Trityl Assay

  • 0.1 M para‐toluene sulfonic acid (TSA; monohydrate) in recipeacetonitrile
  • 15‐ml glass tubes (graduated, if possible)

Support Protocol 1: Using the Trityl Assay for Troubleshooting

  • recipeConcentrated (14.8 N) ammonium hydroxide (see recipe)
  • Triethylamine
  • 3:1 (v/v) recipeconcentrated ammonium hydroxide/ethanol
  • n‐Butanol
  • Screw‐cap plastic vial (preferably fitted with rubber O ring)
  • Heat block or oven, 55° to 60°C
  • 0.2‐µm filter

Basic Protocol 2: Deprotection of DNA Oligonucleotides

  • 100% ethanol
  • 3:1 (v/v) recipeconcentrated (14.8 N) ammonium hydroxide/ethanol
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • Triethylamine trihydrofluoride
  • Screw‐cap plastic vial (preferably fitted with rubber O ring)
  • Heat block or oven, 55° to 60°C
  • 0.2‐µm filter
  • Sephadex G‐25 column (Amersham Pharmacia Biotech)
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Literature Cited

Literature Cited
  Applied Biosystems. 1988. User Bulletin no. 13. Applied Biosystems, Foster City, Calif.
   Bartel, D. and Szostak, J.W. 1993. Isolation of new ribozymes from a pool of random sequences. Science 261:1411‐1418.
   Boal, J.H., Wilk, A., Harindranath, N., Max, E.E., Kempe, T., and Beaucage, S.L. 1996. Cleavage of oligodeoxyribonucleotides from controlled‐pore glass supports and their rapid deprotection by gaseous amines. Nucl. Acids Res. 24:3115‐3117.
   Ciccarelli, R.B., Gunyuzlu, P., Huang, J., Scott, C., and Oakes, F.T. 1991. Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands. Nucl. Acids Res. 19:6007‐6013.
   Cohen, G., Deutsch, J., Fineberg, J., and Levine, A. 1997. Covalent attachment of DNA oligonucleotides to glass. Nucl. Acids Res. 25:911‐912.
   Derbyshire, K.M., Salvo, J.J., and Grindley, N. 1986. A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene 46:145‐152.
   Eadie, J.S., McBride, L.J., Efcavitch, J.W., Hoff, L.B., and Cathcart, R. 1987. High‐performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition. Anal. Biochem. 165:442‐447.
   Farrance, I.K., Eadie, J.S., and Ivarie, R. 1989. Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer. Nucl. Acids Res. 17:1231‐1245.
  Gait, M.J. (ed.). 1984. Oligonucleotide Synthesis: A Practical Approach. IRL Press, Washington D.C..
   Gillam, S. and Smith, M. 1980. Use of E. coli polynucleotide phosphorylase for the synthesis of oligodeoxyribonucleotides of defined sequence. Methods Enzymol. 65:687‐701.
   Hermes, J.D., Parekh, S.M., Blacklow, S.C., Kuster, H., and Knowles, J.R. 1989. A reliable method for random mutagenesis: The generation of mutant libraries using spiked deoxyribonucleotide primers. Gene 184:143‐151.
   Horn, T. and Urdea, M.S. 1988. Solid supported hydrolysis of apurinic sites in synthetic oligonucleotides for rapid and efficient purification on reverse‐phase cartridges. Nucl. Acids Res. 16:11559‐11571.
   Kawahara, S., Wada. T., and Sekine, M. 1996. Unprecedented mild acid‐catalyzed desilyation of the 2‐O‐tert ‐butyldimethylsilyl group from chemically synthesized oligoribonucleotides intermediates via neighboring group participation of the internucleotide phosphate residue. J. Am. Chem. Soc. 118:9461‐9468.
   Kinoshita, Y., Nishigaki, K., and Husimi, Y. 1997. Fluorescence‐, isotope‐ or biotin‐labeling of the 5′‐end of single‐stranded DNA/RNA using T4 RNA ligase. Nucl. Acids Res. 25:3747‐3748.
   Oliphant, R. 1989. Functional Sequences from Random DNA. Harvard University Thesis, Boston, Mass.
   Oliphant, R., Nussbaum, A.L., and Struhl, K. 1986. Cloning of random‐sequence oligodeoxynucleotides. Gene 44:177‐183.
  Pharmacia. 1989. Analects Vol. 17, no. 2. Pharmacia Biotech, Piscataway, NJ.
   Ratliff, R.L. 1982. Terminal deoxynucleotidyltransferase. In The Enzymes, Vol. XV (P.D. Boyer, ed.) pp. 105‐118. Academic Press, San Diego.
   Reddy, M.P., Hanna, N.B., and Farooqui, F. 1994. Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett. 35:4311‐4314.
   Reddy, M.P., Farooqui, F., Hanna, N.B. 1995. Methylamine deprotection provides increased yield of oligoribonucleotides. Tetrahedron Lett. 36:8929‐8932.
   Reidhaar‐Olson, J.F. and Sauer, R.T. 1988. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science 241:53‐57.
   Schulhof, J.C., Molko, D., and Teoule, R. 1987. The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base‐protecting groups. Nucl. Acids Res. 15:397.
   Sproat, B., Colonna, F., Mullah, B., Tsou, D., Andrus, A., Hampel, A., and Vinayak, R. 1995. An efficient method for the isolation and purification of oligoribonucleotides. Nucleosides Nucleotides 14:255‐273.
   Szostak, J. 1992. In vitro genetics. Trends Biochem. Sci. 17:89‐93.
   Tanaka, T. and Letsinger, R.L. 1982. Syringe method for stepwise chemical synthesis of oligonucleotides. Nucl. Acids Res. 10:3249.
   Uhlenbeck, O.C. and Gumport, R.I. 1982. T4 RNA ligase. In The Enzymes, Vol. XV (P.D. Boyer, ed.) pp. 31‐58. Academic Press, San Diego.
   Usman, N., Ogilvie, K.K., Jiang, M.Y., and Cederagren, R.J. 1987. Automated chemical synthesis of long oligoribonucleotides using 2′‐O ‐silylated ribonucleotide 3′‐O ‐phosphoramidites on a controlled‐pore glass support: Synthesis of a 43‐nucleotide sequence similar to the 3′ half molecule of an E. coli formylmethionine tRNA. J. Am. Chem. Soc. 109:7845‐7854.
   Vargeese, C., Carter, J., Yegge, J., Karivjansky, S., Settle, A., Kropp, E., Peterson, K., and Peiken, W. 1998. Efficient activation of nucleoside phosphoramidites with 4,5‐dicyanoimidazole during oligonucleotide synthesis. Nucl. Acids Res. 26:1046‐1050.
   Warren, W.J. and Vella, G. 1994. Analysis and purification of synthetic oligonucleotides by high‐performance liquid chromatography. Methods Mol. Biol. 233‐264.
   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucl. Acids Res. 23:2677‐2684.
   Zhu, Y., He, L., Srinivasan, R., and Lubman, D. 1997. Improved resolution in the detection of oligonucleotides up to 60‐mers in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry using pulsed‐delayed extraction with a simple high voltage transistor switch. Rapid Commun. Mass Spectrom. 11:987‐992.
   Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Summers, M.F., and Byrd, R.A. 1985. Analytical studies of “mixed sequence” oligodeoxyribonucleotides synthesized by competitive coupling of either methyl or β‐cyanoethyl‐N,N‐diisopropylamino phosphoramidite reagents, including 2′‐deoxyinosine. Nucl. Acids Res. 13:8181‐8196.
Key References
   Applied Biosystems, 1988. See above.
   A well‐organized overview of synthetic oligonucleotide synthesis, purification, and quantitation.
   Bretherick, L., 1986. Hazards in the Chemical Laboratory, 4th ed. Alden Press, Oxford.
  A guide to hazardous chemical handling.
   Gait, 1986. See above.
  The seminal text on synthetic oligonucleotide synthesis that provides critical insight.
Internet Resources
  Web site detailing synthesis chemistries, procedures, and reagents for solid‐phase oligonucleotide chemistry.
  Web site providing general advice on oligonucleotide synthesis.
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