Cycle

Etc.

Reagents

In the most common method of making DNA, phosphoramidite groups react to link nucleotides together.

DNA. The first problem is that each deoxynucleotide has two hydroxyl groups, one used for bonding to the nucleotide ahead of it and the other for bonding to the nucleotide behind it in the nucleic acid chain. Chemical reagents cannot distinguish between these two hydroxyls. Therefore, each time a nucleotide is added, one of its hydroxyl groups must first be chemically blocked and the other must be activated. The standard phosphoramidite method for artificial chemical synthesis of DNA proceeds in the 3' to 5' direction. Consequently, before adding any new nucleotide to the chain, the 5'-hydroxyl of the previous nucleotide is blocked with a dimethoxytrityl (DMT) group and the 3'-hydroxyl is activated with a phosphoramidite. Note that the reagents for chemical synthesis are phosphoramidite nucleotides (with a single phosphorus), not nucleoside triphosphates as in biosynthesis (Fig. 21.11). Also, chemical synthesis occurs in the 3' to 5' direction, the opposite of biological DNA synthesis, which always occurs 5' to 3'.

The first nucleotide is anchored to a glass bead via its 3'-OH group. The first base is actually added as a nucleoside without any phosphate group. It is bound to the glass bead via a spacer molecule (Fig. 21.12). The spacer helps prevent the bases in the growing nucleotide chain from reacting with the glass bead surface.

Next, acid (often trichloroacetic acid, TCA) is poured through the column to remove the DMT blocking group and expose the 5'-hydroxyl group on the first nucleotide. The second phosphoramidite nucleotide is added to the column. This nucleotide links to the first one via the single phosphate in the phosphoramidite moiety (Fig. 22.13). After each reaction step, the column is washed by acetonitrile to remove unreacted reagents and then flushed with argon to remove any traces of acetonitrile. The cycle continues until a full-length sequence is manufactured.

dimethoxytrityl (DMT) group Group used for blocking the 5'-hydroxyl of nucleotides during artificial DNA synthesis phosphoramidite method Method for artificial synthesis of DNA that utilizes the reactive phosphoramidite group to make linkages between nucleotides

FIGURE 21.10 DNA Synthesizer

Biologist programs an automated DNA synthesizer to produce a specific oligonucleotide for her research. Credit: Hank Morgan, Photo Researchers, Inc.

FIGURE 21.10 DNA Synthesizer

Biologist programs an automated DNA synthesizer to produce a specific oligonucleotide for her research. Credit: Hank Morgan, Photo Researchers, Inc.

Diisopropylamino group

FIGURE 21.11 Phosphoramidite Nucleotides are used for Chemical Synthesis of DNA

Diisopropylamino group

FIGURE 21.11 Phosphoramidite Nucleotides are used for Chemical Synthesis of DNA

During chemical synthesis of DNA, modifications must be added to each nucleotide to ensure that the correct group reacts with the next chemical reagent. Each nucleotide has a blocking DMT group attached to the 5'-OH. The 3'-OH is activated by attaching a phosphoramidite group.

FIGURE 21.12 Addition of Spacer Molecule and First Base to Glass Bead

The first nucleotide is linked to a glass bead via a spacer molecule attached to its 3'-OH group.

Initial 1 nucleotide

Initial 1 nucleotide

FIGURE 21.12 Addition of Spacer Molecule and First Base to Glass Bead

The first nucleotide is linked to a glass bead via a spacer molecule attached to its 3'-OH group.

Second nucleotide

Activating group

Activating group

Blocking group

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