1. Chapter 5 Chemical Synthesis, Sequencing, and Amplification of DNA

2. 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

3. 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.

4. Bases

5. 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

6. 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

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

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

9. This cycling continues. (Fig. 5.1)

10. 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

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

12. 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)

13. Fig. 5.10

14. Fig. 5.11

15. 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

16. Fig. 5.12

17. Fig. 5.13