Introduction

Deoxyribonucleic acid (DNA) synthesis is a process by which copies of nucleic acid strands are made, and till now, Oligonucleotide synthesis is one of the most widely used technology to synthesis relatively short fragments of DNA. Opposite of biosynthesis, the Oligonucleotide synthesis proceeds in the 3’→5’ direction.

The method includes four different steps:
1. Detritylation (de-blocking): The synthesis cycle initiated by removing the 5’-DMT protecting group of the solid-support-linked nucleoside, the step results in the solid support-bound oligonucleotide precursor bearing a free 5’-terminal hydroxyl group.
2. Coupling: Once the DMT has been removed, the free 5’-OH of the solid-support-linked nucleoside is able to react with the next nucleotide, which is a phosphoramidite monomer.
3. Capping: The capping step is performed by treating the solid support-bound material with a mixture of acetic anhydride and 1-methylimidazole. This step could eliminate the un-reacted 5’-OH react during the next cycle.
4. Oxidation: The phosphite triester formed in this cycle is unnatural and unstable. Therefore, we treat the support-bound material with iodine and water in the presence of a weak base to oxidize the phosphite triester into a tetra-coordinated phosphate triester. This step could convert the phosphite triester to the stable phosphate triester.

After oxidation, the synthesis gets into the second cycle. The number of cycles repeated equals the desired number of bases.

Fig. 1 DNA synthesis steps

Polyacrylamide gel electrophoresis is a powerful purification method for DNA. In this technology, DNA was separated by electrophoresis first, the wanted DNA band was visualized and cut out from the gel. The slice which contained DNA then soaked briefly in water to remove gel buffer and ethidium bromide.

Fig. 2 Methods of DNA/RNA Purification from PAGE Gels.

The DNA molecules were recovered by running another electrophoresis, which causes the band to migrate out of the matrix and into the surrounding buffer. Thus we got high purity and high recovery DNA molecules.

Procedures:
1. DNA sequence optimization and oligo design.
2. Fragments assembly.
3. DNA clone.
4. DNA screening.
5. DNA sequencing check.

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