Oligonucleotides Synthesis

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The Chemical Synthesis of Oligonucleotides and the Phosphoramidite Method

By Klaus D. Linse

The study of nucleic acids has now become a fruitful and dynamic scientific enterprise. Nucleic acids are of unique importance in biological systems. Genes are made up of deoxyribonucleic acid or DNA, and each gene is a linear segment, or polymer, of a long DNA molecule. A DNA polymer, or DNA oligonucleotide, contains a linear arrangement of subunits called nucleotides. There are four types of nucleotides. Each nucleotide has three components; a phosphate group, a sugar and a base that contains nitrogen within its structure. The sugar moiety in DNA oligonucleotides is always dexoyribose, and there are four alternative bases: adenine (A), thymine (T), guanine (G), and cytosine (C). The phosphate groups and the deoxyribose sugars form the backbone of each DNA stand. The bases are joined to the deoxyribose sugar and stick out to the side. Both oligomers, DNA and RNA, consist of 5’->3’ phosphodiester-linked nucleotide units that are composed of a 2’-deoxy-D-ribose (DNA) or D-ribose (RNA) in their furanose forms and a heteroaromatic nucleobase (A, T, G, and C; A, U, G, C), and the resulting oligonucleotide chain is composed of a polar, negatively charged sugar-phosphate backbone and an array of hydrophobic nucleobases. The amphiphilic nature of these polymers dictates the assembly and maintenance of secondary and tertiary structures the oligonucleotides can form. In the DNA duplex structure, genetic information is stored as a linear nucleotide code. This code can be accessed and replicated. RNA, or ribonucleic acid, is another structurally related essential biopolymer. RNA differs from DNA in having the sugar ribose in place of the deoxyribos...

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...oligomer, possible truncated failure sequences with free 5’hydroxyl ends, byproducts of deprotection, and silicates from hydrolysis of the glass support. Different purification methods can be used to separate the product oligonucleotide from the contaminating species.

Figure 18: summary of Phosphoamidite Oligonucleotide Synthesis Method.

Reference

J.C. Biro, B. Benyó, C. Sansom, Á. Szlávecz, G. Fördös, T. Micsik, and Z. Benyó; A common periodic table of codons and amino acids. Biochemical and Biophysical Research Communications 306 (2003) 408–415.

Oligonucleotides and analogues: A practical approach. Edited by F Eckstein. Series editors: D. Rickwood and B.D. Hames. IRL PRESS. Oxford University Press. 1991.

Operation Manual. MilliGen/Biosearch Cyclone™ Plus DNA Synthesizer.

Winnacker, Ernst L.; From genes to clones: introduction to gene technology. VCH, 1987.

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