Molecular Biotechnology. Bernard R. Glick

of viral RNA and proteins that were assembled into infectious poliovirus particles.

      Construction of a much larger synthetic bacterial genome presented a greater challenge (Gibson et al., Science. 319:1215–20, 2008). The genome of the bacterium Mycoplasma genitalium was chosen because it has the smallest genome (a single chromosome of 580,076 bp) currently known for a free-living bacterium. The chromosome was initially produced in 101 segments of about 5 to 7 kb that were each assembled from synthetic oligonucleotides. The sequence of each segment overlapped its neighbor by approximately 80 bp and therefore, following brief treatment with an exonuclease to generate sticky ends, four neighboring segments were joined in vitro by complementary base-pairing at their termini and enzymatic repair of gaps. These 24-kb fragments were cloned into a bacterial artificial chromosome, which can carry large DNA inserts, and propagated in E. coli. This in vitro recombination method was repeated to assemble the fragments into successively larger pieces. Large segments carrying half- and full-genome sequences could not be cloned in E. coli and therefore quarter- and then half-genome fragments were finally assembled into a 582,970 bp sequence in the yeast Saccharomyces cerevisiae by in vivo recombination between overlapping homologous sequences.

      These results demonstrated that a small bacterial genome can be constructed entirely from synthetic oligonucleotides. The next step was to show that a genome produced “from scratch” can direct the survival and growth of a living bacterium. The 1,077,947 bp genome of Mycoplasma mycoides was synthesized in a manner similar to that used to construct the M. genitalium genome (Gibson et al., Science 329:52–56, 2010); M. mycoides was chosen because it grows at a faster rate than the extremely slow growing M. genitalium. Briefly, overlapping synthetic oligonucleotides were assembled into 1,080 bp fragments. These fragments were recombined into 10-kb and then into 100-kb segments, and finally into a full-length genome by in vivo homologous recombination in yeast. The intact M. mycoides genome was extracted from yeast and transplanted into a related species Mycoplasma capricolum, replacing the recipient cell’s chromosome. Remarkably, the synthetic genome was self-replicating and controlled the functions of a living bacterium. The bacterium was able to grow logarithmically and exhibited cellular and colony morphologies similar to M. mycoides. “Watermark” sequences were inserted in four regions to enable differentiation of the synthetic and natural genomes.

      A major motivation for producing a synthetic bacterium is to understand the minimal genetic requirements for life, that is, to identify the minimum set of essential genes that can support survival and reproduction of a cell. Because it takes less energy and fewer resources to maintain and propagate a small genome, more resources can be directed to the synthesis of high yields of useful products from cloned genes.

Synthesis of Oligonucleotides

      Currently, the phosphoramidite method is the procedure of choice for chemical DNA synthesis. Solid-phase synthesis, in which the growing DNA strand is attached to a solid support, is used so that all the reactions can be conducted in one reaction vessel, the reagents from one reaction step can be readily washed away before the reagents for the next step are added, and the reagents can be used in excess in an attempt to drive the reactions to completion.

      The chemical synthesis of DNA is a multistep process (Fig. 2.25). It does not follow the biological direction of DNA synthesis; rather, during the chemical process, each incoming nucleotide is coupled to the 5′ hydroxyl terminus of the growing chain. Before their introduction into the reaction column, the amino groups of the nucleotides’ nitrogenous bases adenine, guanine, and cytosine are derivatized by the addition of benzoyl, isobutyryl, and benzoyl groups, respectively, to prevent undesirable side reactions during polymerization. Thymine is not treated because it lacks an amino group. The initial nucleoside (base and sugar only), which will be the 3′-terminal nucleotide of the final synthesized strand, is attached to a spacer molecule by its 3′ hydroxyl terminus and the spacer molecule is covalently attached to an inert support, which is often a controlled pore glass (CPG) bead (a glass bead with uniformly sized pores) (Fig. 2.26). A dimethoxytrityl (DMT) group is attached to the 5′ end of the first nucleoside to prevent the 5′ hydroxyl group from reacting nonspecifically before the addition of the second nucleotide. Each nucleotide that is added to the growing chain has a 5′ DMT protective group and also a diisopropylamine group attached to a 3′ phosphite group that is protected by a β-cyanoethyl (CH2CH2CN) group (Fig. 2.27). This molecular assembly is called a phosphoramidite.

      Figure 2.25 Flowchart for the chemical synthesis of DNA oligonucleotides. After n coupling reactions (cycles), a single-stranded piece of DNA with n + 1 nucleotides is produced.

      Figure 2.26 Starting complex for the chemical synthesis of a DNA strand. The initial nucleoside has a DMT group attached to the 5′ hydroxyl group of the deoxyribose moiety and a spacer molecule attached to the hydroxyl group of the 3′ carbon of the deoxyribose. The spacer unit is attached to a solid support, which is usually a CPG bead.

      Figure 2.27 Structure of a phosphoramidite. Phosphoramidites are available for each of the four bases (A, C, G, and T) that are used for the chemical synthesis of a DNA strand. A diisopropylamine group is attached to the 3′ phosphite group of the nucleoside. A β-cyanoethyl (CH2CH2CN) group protects the 3′ phosphite group, and a DMT group is bound to the 5′ hydroxyl group of the deoxyribose sugar.

      After the first nucleoside is bound to the CPG beads, the cycle begins. First, the reaction column is washed extensively with an anhydrous reagent (e.g., acetonitrile) to remove water and any nucleophiles that may be present. The column is flushed with argon to purge the acetonitrile. Next, the 5′ DMT group is removed from the attached nucleoside by treatment with trichloroacetic acid (TCA) to yield a reactive 5′ hydroxyl group (Fig. 2.28). After this detritylation step, the reaction column is washed with acetonitrile to remove the TCA and then with argon to remove the acetonitrile. The machine is programmed to introduce the next prescribed base (phosphoramidite) and tetrazole simultaneously for the activation and coupling steps. The tetrazole activates the phosphoramidite so that its 3′ phosphite forms a covalent bond with the 5′ hydroxyl group of the initial nucleoside (Fig. 2.29). Unincorporated phosphoramidite and tetrazole are removed by flushing the column with argon.

      Figure 2.28 Detritylation. The 5′ DMT group is removed by treatment with TCA. In this example, the detritylation of the first nucleoside is depicted.

      Figure 2.29 Activation and coupling. The activation of a phosphoramidite enables its 3′ phosphite group to attach to the 5′ hydroxyl group of the bound detritylated nucleoside.

      Not all of the support-bound nucleosides are linked to a phosphoramidite during the first coupling reaction, and therefore, the unlinked residues must be prevented from linking to the next nucleotide during the following cycle. To do this, acetic anhydride and dimethylaminopyridine are added to acetylate the unreacted 5′ hydroxyl groups (Fig. 2.30). If this capping step is not carried out, then, after a number of cycles, the growing chains will differ in both length and nucleotide sequence.