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Overview of Purification and Analysis of Synthetic Nucleic Acids

Alex Andrus¹, Robert G. Kuimelis²

¹PE Applied Biosystems, Foster City, California
²Phylos, Inc., Lexington, Massachusetts

Publication Name:

Current Protocols in Nucleic Acid Chemistry

Unit Number:

Unit 10.3

DOI:

10.1002/0471142700.nc1003s01

Online Posting Date:

May, 2001

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Alex Andrus

Abstract

Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

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Unit Introduction
Selecting Analysis/Purification Methods
Post-Synthesis Procedures for Oligonucleotides
Mixed-Base Oligonucleotides
Modified Oligonucleotides
Literature Cited
Figures
Tables

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Figures

Figure 10.3.1

Absorbance spectra of four natural deoxynucleosides, dA, dC, dG, and T in aqueous solution at pH of 7.0.

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Figure 10.3.2

Absorbance spectra of an 18-nt oligonucleotide 5¢ TCA CAG TCT GAT CTC GAT 3¢ in 0.1 M TEAA, pH 7.0.

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Literature Cited

Literature Cited
	Andrus, A. 1992a. AutoAnalysis: Trityl monitoring of DNA synthesizer operation by conductivity. Research News, Applied Biosystems, Foster City, California.
	Andrus, A. 1992b. Evaluating and Isolating Synthetic Oligodeoxynucleotides. Applied Biosystems, Foster City, California.
	Andrus, A. 1992c. Oligodeoxynucleotide analysis by gel capillary electrophoresis. In Methods: A Companion to Methods in Enzymology, Vol. 4 (J. Wiktorowicz ed.) pp. 213-226. Academic Press, San Diego.
	Andrus, A. 1994. Gel-capillary electrophoresis analysis of oligonucleotides. In Protocols for Oligonucleotide Conjugates (S. Agrawal, ed.) pp. 277-300. Humana Press, Totowa, N.J.
	Beaucage, S.L. and Iyer, R.P. 1992. Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223-2311.
	Bellenson, J. and Smith, A.J. 1992. Increasing DNA synthesizer throughput via off-instrument cleavage and deprotection. BioTechniques 12:219-220.
	Boal, J.H., Wilk, A., Harindranath, N., Max, E., Kempe, T., and Beaucage, S.L. 1996. Cleavage of oligodeoxyribonucleotides from controlled-pore glass supports and their rapid deprotection by gaseous amines. Nucleic. Acids Res. 24:3115-17.
	Caruthers, M.H. and Beaucage, S.L. 1983. Phosphoramidite compounds and processes. United States Patent 4,415,732.
	Caruthers, M.H. and Matteucci, M.D. 1984. Process for preparing polynucleotides. United States Patent 4,458,066.
	Ellington, A. 1995. Synthesis and purification of oligonucleotides. In Current Protocols in Molecular Biology, Vol.1 John Wiley & Sons (F. Ausubel et al, eds.) Unit 2.11., New York.
	Kaufman, J., Le, M., Ross, G., Hing, P., Budiansky, M., Yu, E., Campbell, E., Yoshimura, V., Fitzpatrick, V., Nadimi, K., and Andrus, A. 1993. Trityl monitoring of automated DNA synthesizer operation by conductivity: A new method of real-time analysis. BioTechniques 14:834-839.
	McCollum, C. and Andrus, A. 1991. An optimized polystyrene support for rapid, efficient oligonucleotide synthesis. Tetrahedron Lett. 32:4069-4072.
	Reynolds, T.R. and Buck, G.A. 1992. Rapid deprotection of synthetic oligonucleotides. BioTechniques 12:518-521.
	Theisen, P., McCollum, C., and Andrus, A. 1993. N-6-Dialkylformamidine-2¢-deoxyadenosine phosphoramidites in oligodeoxynucleotide synthesis. Rapid deprotection of oligodeoxynucleotides. Nucleosides Nucleotides 12:1033-1046.