Chemical synthesis of DNA is usually done with an automated synthesizer that creates DNA by sequentially adding one nucleotide after another in the correct sequence order. The first step is attaching the first nucleotide to a porous support material. The pores of support materials allow reagents to be washed through and removed easily. Commonly used support materials are controlled-pore glass (CPG) and polystyrene, which are packaged into columns whose scale depends on both the quantity and the length of oligonucleotide desired. Polystyrene or large-pore CPG columns are recommended for oligonucleotides greater than 50 bases in length. Since CPG does not swell or contract in various solvents and possesses mechanical properties that offer distinct advantages as a polymer support for synthesis. In addition, it gives faster and more efficient coupling than other polymer supports. The most used CPG supports have pores of 500 Å and 1000 Å.
In the chemical synthesis of DNA by phosphoramidite methods, a protective strategy should be adopted to cover up the functional groups on the phosphoramidite monomer. In traditional protection schemes, the nucleophilic amino moieties on the bases are protected with either isobutyryl (iBu) or benzoyl (Bz) groups, both of which can be removed at the completion of synthesis by ammoniolysis. Recent advances have led to the widespread use of phenoxyacetyl (PAC) protection of adenosine, dimethylformadine (DMF) protection of guanosine, and acyl protection of cytosine to yield oligonucleotides that can be deprotected rapidly under mild conditions. The 5′ primary hydroxyl of the ribose is protected with a dimethoxytrityl (DMT) ether moiety which is removed by mild protic acids at the beginning of each coupling cycle. The convenience of using these protecting groups for nucleic acid synthesis is that they yield nearly lesion-free natural DNA with high efficiency through simple hydrolysis, nucleophilic displacement, and redox chemistries. In a standard synthesis cycle, the nucleotide chain grows from an initial protected nucleoside derivatized via its terminal 3′ hydroxyl to a solid support. Assembly of the protected oligonucleotide chain is carried out in four chemical steps, including deblocking, activation/coupling, oxidation, and capping. After cleavage and deprotection, the desired single-stranded nucleic acids are obtained.
Amerigo Scientific offers a wide range of DNA phosphoramidites with protecting groups, support materials, and columns to meet various needs of natural DNA synthesis.
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