Oligonucleotide synthesis the phosphoramidite method

The ability to synthesize chemically short sequences of single-stranded DNA (oligonucleotides) is an essential part of many aspects of genetic engineering. The method most frequently employed is that of solid-phase synthesis, where the basic philosophy is the same as that in solid-phase peptide synthesis (see Section 13.6.3). In other words, the growing nucleic acid is attached to a suitable solid support, protected nucleotides are supplied in the appropriate sequence, and each addition is followed by repeated coupling and deprotection cycles. 

As with peptide synthesis, similar considerations must to be incorporated into the methodology. Vulnerable functional groups in the base, the sugar. 

In solid-phase syntheses, oligonucleotides are usually synthesized in the 5 -direction from an immobilized 3 -terminus. The solid phase is generally silica or controlled pore glass (CPG), which has been 

The bases adenine, guanine, and cytosine all contain exocyclic amino substituents that require protection, since these are potential nucleophiles. They are converted into amides that are stable to the other reagents used in the process, yet can be removed readily by basic hydrolysis. The most effective protecting groups have been found to be isobutyryl for the amino group of guanine, and benzoyl for adenine and cytosine. Thymine has no exocyclic nitrogen and does not need protection. 

Protection and activation of the phosphate moiety is achieved by employing a phosphoramidite derivative, -P(OR)NR2. This reagent has phosphorus in its P oxidation state the phosphate that we finally require contains P. Favoured R groups in the 

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