One of the experimental techniques that played a key part in the rapid development of genetic engineering is the chemical synthesis of oligonucleotides of defined structure. The chemistry of deoxyoligonucleotide synthesis has been the subject of study for many years. The oligonucleotides of any specified sequence can be synthesized by a series of chemical reactions. Solid phase synthesis methods have made the construction of short, defined sequence DNA fragments accessible to all workers and it is often an essential part of the process of isolating a single gene from libraries containing tens of thousands to millions of gene fragments. Currently, the phosphoramidite method of chemical DNA synthesis is the procedure of choice. Chemical synthesis takes place in the 3'-5' direction (reverse of the biological polymerization direction). The multi-step synthesis proceeds as described below:
1. Columns containing a first immobilized nucleotide
All solid phase method cycles start with one nucleoside immobilized via its 3' -OH group and the coupling of the next nucleoside to the 5' -OH group of the first. The 3' -OH group of sugar is covalently attached to an insoluble and inert resin or matrix, typically either controlled pore glass or silica bead, with a long alkyl amine spacer chain. The growing chain remains bound during the reaction series. The chain is thus synthesized from 3'-5' end. The eventual product has free OH groups at both its 3' and 5' ends.
2. Protection of reactive 5' -OH and amine groups
All the incoming nucleotides must be blocked at 5' -OH. The 5' -OH protecting groups are common to all synthetic strategies. All the reactions for the synthesis cycle start with the nucleoside blocked at the 5' carbon by a protecting dimethoxytrityl (DMTr) group and amino groups protected with N-benzoyl or N-isobutyryl derivatives.
The experimental conditions which result in the formation of a phosphodiester link between nucleosides, will lead to undesirable side reactions unless we block them. The exocyclic amino groups on three of the four bases (adenine, guanine and cytosine) require protection, as do the free OH groups on the phosphates.
3. Deprotection or Detritylation
The 5' -OH protecting group must be removed prior to each nucleoside addition. The DMTr is rapidly removed by protic acids, such as trichloroacetic acid or dichloroacetic acid. The intense orange color of the dimethoxytrityl cation released allows the estimation of the amount of nucleoside deprotected.
4. Coupling reaction
The efficiency of the coupling step is critical to the success of a synthesis; that is, the coupling of the activated 3' phosphoramidite to the deprotected 5' ends of the oligonucleotide. In the coupling step, the second base is added in the form of a nucleoside phosphoramidite derivative whose 5' -OH bears a dMTr blocking group so it cannot polymerize with itself. The presence of a weak acid, such as tetrazole, activates the phosphoramidite, and it rapidly reacts with the free 5'-OH of base 1, forming a dinucleotide linked by a phosphite group. Chemical synthesis thus takes place in the 3'-5' direction.
This involves the re-blocking of any oligonucleotide 5' ends that were not reacted with the phosphoramidites. The unreacted free 5'- OHs of base 1 are blocked from further participation in the polymerization process by acetylation with acetic anhydride, referred to as capping.
6. Oxidation of the phosphorus to the pentavalent state
The phosphate linkage between base 1 and base 2 is highly reactive and it is oxidized by iodine water or aqueous iodine (I2) to form the desired more stable phosphate group. This step completes the cycle.
7. Cycle repetition
This consists of repetition of above mentioned steps, until the desired full length oligonucleotide is achieved.
This is an alkaline treatment to achieve cleavage of the cyanoethyl groups attached to the phosphodiester bonds, linking the individual nucleotides and hydrolysis of the terminal 3' ester bond, to release the oligonucleotide. When the chain is complete, it is cleaved from the support with NH4OH, which also removes the N-benzoyl and N-isobutyryl protecting groups from the amino functions on the A, G and C residues.
The newly synthesized oligonucleotide product is present in a mixture of prematurely terminated chains and cleaved chains. The freed oligonucleotides can be purified by gel electrophoresis, or by High Pressure Liquid Chromatography, Reverse Phase- High Pressure Liquid Chromatography, etc.
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