Molecular Biotechnology. Bernard R. Glick

      Figure 2.30 Capping. The available 5′ hydroxyl groups of unreacted detritylated nucleosides are acetylated to prevent them from participating in the coupling reaction of the next cycle.

      At this stage of the process, the linkage between the nucleotides is in the form of a phosphite triester bond, which is unstable and prone to breakage in the presence of either acid or base. Therefore, the phosphite triester is oxidized with an iodine mixture to form the more stable pentavalent phosphate triester (Fig. 2.31). After this oxidation step and a subsequent wash of the reaction column, the cycle of detritylation, phosphoramidite activation, coupling, capping, and oxidation is repeated (Fig. 2.25). This cycling continues with each successive phosphoramidite until the last programmed residue has been added to the growing chain. When the final cycle is completed, the newly synthesized DNA strands are bound to the CPG beads; each phosphate triester contains a β-cyanoethyl group; every guanine, cytosine, and adenine carries its amino-protecting group; and the 5′ terminus of the last nucleotide has a DMT group.

      Figure 2.31 Oxidation. The phosphite triester internucleotide linkage is oxidized to the pentavalent phosphate triester. This reaction stabilizes the phosphodiester bond and makes it less susceptible to cleavage under either acidic or basic conditions.

      The β-cyanoethyl groups are removed by a chemical treatment in the reaction column. The DNA strands are then cleaved from the spacer molecule leaving a 3′ hydroxyl terminus. The DNA is eluted from the reaction column, and, in succession, the benzoyl and isobutyryl groups are stripped away and the DNA is detritylated. The 5′ terminus of the DNA strand is phosphorylated either enzymatically with T4 polynucleotide kinase or by a chemical procedure. Phosphorylation can also be carried out after detritylation while the oligonucleotide is still bound to the support.

      To achieve a reasonable overall yield of an oligonucleotide, the coupling efficiency should be greater than 98% at each step. The coupling efficiency of each cycle is determined by spectrophotometrically monitoring released trityl groups. If, for example, the efficiency is 99% at each cycle during the production of a 20-unit oligonucleotide (20-mer), which entails 19 coupling reactions since the first base is bound to the spacer and is not involved in a coupling step, then 83% (i.e., 0.9919 × 100) of the product will be 20 nucleotides long. If a 60-mer is synthesized with 99% efficiency at each cycle, then about 55% of the final product will contain all of the 60 nucleotides. With an average coupling efficiency consistently less than 98%, the yield of full-length oligonucleotides diminishes as a function of the required number of cycles (Table 2.3). The coupling efficiency for most commercial DNA synthesizers averages about 99.5% for each step. However, depending on the length and stringency of the end use of an oligonucleotide, it may be necessary to purify the final product using either reverse-phase high-pressure liquid chromatography or gel electrophoresis. These methods separate the longer target oligonucleotides from the shorter “failure” sequences.

Table 2.3 Overall yields of chemically synthesized oligonucleotides with different coupling efficiencies

Assembling Oligonucleotides into Genes

      Oligonucleotides are the key components for assembling genes. There are a number of applications for synthetic genes including large-scale production of proteins, testing protein function after changing specific codons, and creating nucleotide sequences that encode proteins with novel properties. The production of short fragments of double-stranded DNA (less than 100 bp) is technically straightforward and can be accomplished by synthesizing two complementary oligonucleotides and then annealing them. For the production of longer DNA molecules such as entire genes, special strategies must be devised because the coupling efficiency of each cycle during chemical DNA synthesis is never 100%. For example, if a gene contains 1,000 bp and the average coupling efficiency is 99.5%, then the proportion of full-length single DNA strands after the last cycle is a minuscule 0.007%. To overcome this problem, synthetic genes are produced from oligonucleotides that are enzymatically assembled into larger double-stranded DNA molecules.

      One method for building a synthetic gene utilizes a set of overlapping oligonucleotides that are about 60 nucleotides in length with approximately 20-base overlaps (Fig. 2.32). After complementary 3′ and 5′ extensions are annealed, large gaps remain, but the base-paired regions are both long enough and stable enough to hold the structure together. After all the oligonucleotides are combined, the gaps are filled by enzymatic DNA synthesis with DNA polymerase I (usually from E. coli). This enzyme uses the 3′ hydroxyl groups as replication initiation points and the single-stranded regions as templates. After the enzymatic synthesis is completed, the nicks are sealed with T4 DNA ligase. For larger genes (≥1,000 bp), smaller sections of the gene are first assembled into units of about 500 bases in length and then these are combined with other 500-base units. In turn, these larger kilobase segments are joined together until the entire sequence is completed. Computer programs are available both commercially and freely on the Internet which make it easier to determine the best set of oligonucleotides and overlaps for gene construction as well as allowing the user to select a particular codon usage, change any codon, and designate restriction endonuclease sites at specific locations. Finally, it is absolutely essential that a chemically synthesized gene have the correct sequence of nucleotides. Consequently, small synthetic genes are sequenced directly and, for larger genes, the sequences of each of the 500-base building blocks are determined before assembly.

      Figure 2.32 Assembly and in vitro enzymatic DNA synthesis of a gene. Individual oligonucleotides are synthesized chemically and then hybridized. The sequences of the oligonucleotides are designed to enable them to form a stable molecule with base-paired regions separated by single-stranded regions (gaps). The gaps are filled in by in vitro enzymatic DNA synthesis. The nicks are sealed with T4 DNA ligase.

Gene Synthesis by PCR

      The assembly of a gene by PCR is faster and more economical than filling in overlapping oligonucleotides using DNA polymerase and then sealing the nicks with T4 DNA ligase. One PCR-based protocol for gene construction starts with two overlapping oligonucleotides (A and B), usually about 50 nucleotides long, that represent sequences from the center of the gene (Fig. 2.33). After annealing, these oligonucleotides have recessed 3′ hydroxyl groups that provide a starting point for DNA synthesis during the elongation phase of a PCR cycle. The product is a double-stranded DNA molecule. The PCR cycle (denaturation, oligonucleotide annealing, and extension) is repeated 20 times to maximize the amount of product that is formed. Next, two additional oligonucleotides (C and D) are added to the mixture. Oligonucleotide C overlaps at its 3′ end with the 5′ end of oligonucleotide A and represents the nucleotide sequence of the gene immediately upstream of the oligonucleotide A sequence. Oligonucleotide D overlaps at its 3′ end with the 5′ end of oligonucleotide B and represents the nucleotide sequence of the gene immediately downstream of the oligonucleotide B sequence. After 20 PCR cycles, a double-stranded DNA with a specific sequence order (CABD) is produced.

      Figure 2.33 Gene synthesis by PCR. Overlapping oligonucleotides (A and B) are filled in from the recessed 3′ hydroxyl ends during DNA synthesis. Oligonucleotides (C and D) that are complementary to the ends of the product of the first PCR cycle are added to a sample, overlapping molecules are formed after denaturation and renaturation, and the recessed ends are filled in during DNA synthesis. Next, oligonucleotides