Chapter 5 Chemical Synthesis, Sequencing, and Amplification of DNA

Chemical Synthesis of DNA DNA synthesizer (gene machine) ssDNA (≤ 50 nucleotides) Incoming nucleotide is coupled to the 5’ hydroxyl terminus of the growing chain cf: natural DNA synthesis

The Phosphoramidite Method The amino groups of A, G, C are derivatized by the addition of benzoyl, isobutyryl, and benzoyl groups, respectively. (T does not have amino group.) to prevent side reactions A space molecule is attached to a solid support (controlled pore glass). Figure 5.2 The initial nucleoside (base + sugar) is attached to the spacer molecule by its 3’-terminus. (The initial nucleoside has been protected by 5’-DMT (dimethoxy trityl).) Washing with acetonitrile to remove water and any nucleophiles. Flushing with argon to purge acetonitrile.

Bases

Detritylation (Figure 5.4) 5’-DMT is removed by treatment with TCA (trichloroacetic acid). Washing with --- acetonitrile --- argon Each nucleotide that is to be added to the growing chain has 5’-DMT group and a diisopropylamine group attached to a methylated 3’-phosphite group  phosphoramidite

Activation and coupling (Figure 5.5) [Add the next base (phosphoramidite) and tetrazole] The tetrazole activates the phosphoramidite to form the covalent bond between 3’-phosphite (#2) and 5’-hydroxyl (#1) group. Flushing with argon

Capping (Figure 5.6) add acetic anhydride and dimethylamino pyridine The unreacted 5’-OH groups are acetylated.

Oxidation (Figure 5.7) Washing with an iodine mixture To make a stable phosphodiester bond Washing

This cycling continues. (Fig. 5.1)

Purification of synthesized oligonucleotide Removing oligonucleotide from column Removal of methyl group by a chemical treatment (Fig. 5.7) Cleavage and elution of DNA strands from the spacer Removal of benzoyl and isobutyryl group from each base DMT at 5’-terminus is removed by detritylation, 5’-terminus is phosphorylated by enzymatic or chemical method. Purification by reverse-phase high-pressure liquid chromatography (HPLC) or gel electrophoresis

Uses of Synthesized Oligonucleotides ss probes (20~40 mer) Deduction of codons from the amino acid sequence (Figure 5.8)

Uses of Synthesized Oligonucleotides Linkers Short, double-stranded, blunt-ended oligomer which contains a restriction endonuclease site Figure 5.9: A (6-mer linker) B (8-mer linker) Figure 5.10 Adapter Variant of the linker contains one or more restriction endonuclease sites (Figure 5.9 C, Figure 5.11)

Fig. 5.10

Fig. 5.11

Uses of Synthesized Oligonucleotides Synthesis of double-stranded DNA short gene (60~80 bp) ~ synthesizing the complementary strands and then annealing them longer genes (> 300 bp) ~ Figure 5.12: assembly of a synthetic gene from short oligonucleotides (20- to 60-mer) ~ Figure 5.13: assembly and in vitro enzymatic DNA synthesis of a gene

Fig. 5.12

Fig. 5.13