Molecular Biotechnology: Principles and Applications - Glick, B. R., & Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Chemical Synthesis, Nucleotide Sequencing, and DNA Amplification
Chemical DNA Synthesis

Technological progress in any field of science invariably stimulates its further development. The advent of new technologies makes it possible to conduct novel experiments and facilitates the execution of established ones. The Emergence of molecular BIOTECHNOLOGY AS A science is indebted to a wide array of technological developments; many of these are now widely applied both in large research centers and small scientific teams. Today, it is relatively effortless to chemically synthesize a single DNA molecule, determine The nucleotide sequence of another, and amplify a third using the Polymerase Chain Reaction. All of this has been made possible by the information gathered through foundational research on both DNA itself and The Mechanism of its Replication. These experimental approaches have become an integral part of molecular cloning—a Procedure that enables the isolation of desired DNA fragments, their characterization, and the performance of diverse manipulations upon them.

With The Development of rapid and inexpensive Methods for the Chemical synthesis of single-stranded DNA fragments with a predetermined nucleotide sequence, the methodology of molecular cloning and DNA characterization has changed substantially. Chemically synthesized oligonucleotides can be used to construct entire genes or their fragments, to amplify specific DNA fragments, for Site-Directed Mutagenesis of isolated DNA, as well as Hybridization probes and linkers that facilitate cloning.

With the Introduction of instruments for automated Chemical DNA Synthesis (DNA synthesizers), the PRODUCTION OF SINGLE-stranded oligonucleotides <50 units in length has become a more or less routine procedure. The core component of any DNA synthesizer is a system of Valves and pumps used to introduce NUCLEOTIDES and Reagents into the reaction mixture according to a strictly predetermined program, ensuring the attachment of the required monomer units to the growing chain. Unlike biological synthesis, chemical DNA synthesis allows each new nucleotide to be attached to the 5'-hydroxyl end of the chain. All reactions are carried out sequentially in a single reaction Column, with the duration of each reaction and the washing time controlled by a computer.

The Phosphoramidite Method. Currently, this is the most widespread method of chemical DNA synthesis. Its starting building blocks are modified deoxyribonucleosides. The modification consists of attaching a benzoyl group to the amino groups of deoxyadenosine and deoxycytidine, and an isobutyryl group to the amino group of deoxyguanosine. Thymidine, which lacks an amino group, is not modified. Such modification is necessary to protect the nucleosides from undesirable Side Reactions during chain elongation. The synthesis is carried out in the solid phase (the growing DNA chain is anchored to a solid support), which makes it possible to conduct all reactions in a single vessel, easily wash away unneeded reagents after each step, and add new ones in quantities ensuring the reaction goes to completion as fully as possible.

The steps of the multistep synthesis are illustrated in Fig. 5.1. The first nucleoside (nitrogenous base + sugar) is anchored to an inert solid support, typically porous Glass beads of uniform size. The 3'-hydroxyl group of the first nucleoside, which will serve as the 3'-terminal nucleotide of the synthesized chain, is attached to a spacer molecule covalently linked to the support. To prevent non-specific interactions of the 5'-hydroxyl group of the first nucleotide prior to The addition of the second nucleoside into the reaction mixture, it is protected with a dimethoxytrityl (DMT) group (Fig. 5.2). Every nucleotide attached to the growing chain bears such a group, and additionally carries a diisopropylamine group attached to the 3'-phosphite group, which in turn is protected by a methyl moiety (Fig. 5.3). This molecular configuration is termed a phosphoramidite.

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Fig. 5.1. Chemical synthesis of an oligonucleotide. After n cycles, a single-stranded DNA fragment of n + 1 nucleotides is formed.

Fig. 5.2. Complex initiating the chemical synthesis of a DNA chain. A dimethoxytrityl (DMT) group is attached to the 5'-hydroxyl group of the deoxyribose of the first nucleoside, while a spacer molecule is attached to the 3'-hydroxyl group. The latter, in turn, is linked to a solid support (a porous glass bead).

The cycle begins after the attachment of the first nucleoside to the glass bead. Next, the column is thoroughly washed with an anhydrous reagent (e.g., acetonitrile) to remove Water and other nucleophilic substances, and argon is purged through it to displace the acetonitrile. Then, trichloroacetic acid (TCA) is used to cleave the 5'-DMT group (detritylation) from the attached nucleotide in order to release (expose) the reactive 5'-hydroxyl group (Fig. 5.4). The column is washed again with acetonitrile to remove TCA, and argon is purged through it to remove the acetonitrile. The process is programmed so that In the second step, the next nucleoside (in the form of a phosphoramidite) and tetrazole are simultaneously introduced into the column (activation and coupling). Tetrazole activates the phosphoramidite such that the 3'-phosphite group forms a covalent bond with the 5'-hydroxyl group of the first nucleoside (Fig. 5.5). Unincorporated phosphoramidite and tetrazole are removed by purging with argon.

Fig. 5.3. Structural formula of a phosphoramidite. Such derivatives of all four bases—A, T, G, and C—are used for chemical DNA synthesis. DMT, dimethoxytrityl; Me, methyl group.

Since not all support-bound nucleosides are coupled with the phosphoramidite by the end of the first step, it is necessary to prevent their interaction with the nucleoside added in the second step. To achieve this, unreacted 5'-hydroxyl groups are acetylated using acetic anhydride and dimethylaminopyridine (capping) (Fig. 5.6). If this is not done, the synthesized oligonucleotides will vary in both length and nucleotide sequence after just a few cycles. The phosphite triester bond formed between nucleotides in the second step is unstable and can break in the presence of acids or alkalis. Therefore, the phosphite triester is oxidized with an iodine solution to a more stable pentavalent phosphate triester (Fig. 5.7). The column is then washed, and the entire cycle is repeated (detritylation, activation and coupling, capping, oxidation; Fig. 5.1). These described operations are performed until the final nucleoside is attached to the growing chain According to the program. The synthesized oligonucleotides remain attached to the glass beads; each phosphate triester bears a methyl group; each guanine, cytosine, and adenine contains a protected amino group; and a DMT group is present at the 5'-end of the final nucleotide.

Fig. 5.4. Detritylation—Cleavage of the 5'-dimethoxytrityl (DMT) group using trichloroacetic acid (TCA).

Fig. 5.5. Activation and coupling. The 3'-phosphite group of the activated phosphoramidite forms a covalent bond with the 5'-hydroxyl group of the detritylated nucleoside anchored to the glass bead. DMT, dimethoxytrityl group; Me, methyl group.

Fig. 5.6. Capping. Free 5'-hydroxyl groups of detritylated nucleosides that failed to react in the first cycle are acetylated to prevent their Participation in the next cycle.

Methyl groups are removed by chemical Treatment directly within the reaction column. The oligonucleotides are then detached from the spacer molecule along with the 3'-hydroxyl end and eluted from the column; subsequently, the benzoyl, isobutyryl, and DMT groups are successively removed. The 5'-end of the chain is phosphorylated enzymatically (T4 polynucleotide kinase + ATP) or chemically. This reaction can also be performed while the oligonucleotide is still attached to the support, but following detritylation.

Fig. 5.7. Oxidation. The phosphite triester is oxidized to a pentavalent phosphate triester, which stabilizes the phosphodiester bond and makes it more resistant to the action of acids and alkalis. DMT, dimethoxytrityl group; Me, methyl group.

For the product yield to be sufficiently high, the coupling efficiency of nucleotides at each step must be at least 98%. Efficiency is monitored spectrophotometrically by determining The amount of cleaved trityl groups. If, for instance, the efficiency of each cycle during the synthesis of a 20-mer oligonucleotide is 99%, then 82% (i.e., 0.99²⁰ ∙ 100) of the oligonucleotides will have precisely this length. If a 60-mer oligonucleotide is synthesized at the same efficiency, only 55% of the oligonucleotides will contain 60 nucleotides. Furthermore, if the average cycle efficiency does not exceed 98%, the proportion of oligonucleotides of the desired length will be significantly lower (Table 5.1). Manufacturers of commercial DNA synthesizers typically guarantee an average coupling efficiency of 98%. However, achieving this requires The Use of reagents and chemicals of very high purity, which is not always feasible. As a rule, the actual coupling efficiency is 95%, although 99% efficiency can sometimes be attained. To obtain oligonucleotides of the specified length, the crude products of most chemical syntheses must be purified using either reversed-phase High-Performance Liquid Chromatography or Polyacrylamide gel Electrophoresis. Since all "failed" sequences are shorter than the desired oligonucleotide, this is readily accomplished.

Table 5.1. Average yield of oligonucleotides of specified length (n) at various average cycle efficiencies

Applications of synthesized oligonucleotides

Oligonucleotides synthesized via chemical methods are widely used in molecular biotechnology. They serve as probes in DNA hybridization, as linkers to connect different DNA molecules in cloning experiments, and as primers for DNA Sequencing or site-specific mutagenesis of cloned target genes.

1. The nucleotide sequence of specific oligonucleotide probes (20–40 residues long) is determined based on the Amino Acid Sequence of the corresponding Proteins.

2. To obtain linkers, oligomers are synthesized as palindromic single-stranded nucleotide sequences that hybridize with one another. Linkers contain recognition sites for Restriction Endonucleases, enabling the cloning of DNA fragments (Fig. 5.8, A and B). A short duplex of 6–12 Base Pairs is blunt-end ligated to a DNA target (typically cDNA). The new molecule is then cleaved with the appropriate restriction endonuclease to generate fragments with protruding single-stranded ends (sticky ends), which are used to insert the DNA target into a suitable vector. Prior to insertion, the restricted mixture is fractionated to separate sticky-ended DNA from excess linker molecules. The vector is also treated with a restriction enzyme, annealed with the sticky-ended DNA fragments, and sealed using T4 DNA ligase. The DNA target must not contain restriction sites present in the linker sequence; otherwise, it will also be cleaved by the enzyme.

3. A variation of linker sequences, known as "adapters," often contains sites for two or more restriction endonucleases (Fig. 5.8, C). These allow cDNA to be inserted into a vector via blunt-end ligation and subsequently excised using a different restriction enzyme. The adapter shown in Fig. 5.8, C is inserted into the BamHI site of the vector before cloning the DNA target into the SmaI site by blunt-end ligation. Following cloning, the cDNA is excised from the vector using the BamHI restriction enzyme. In this case, the vector must not contain SmaI sites, and neither the vector nor the cDNA should carry BamHI sites.

Fig. 5.8. Typical linkers and adapter. A. EcoRI linker consisting of 6 base pairs. B. EcoRI linker consisting of 8 base pairs. C. BamHI-SmaI adapter with BamHI sticky ends and a recognition site for SmaI.

4. Single-stranded oligonucleotides of ~17–24 residues are used as primers in DNA sequencing and PCR.

5. Single-stranded oligonucleotides are used as primers for in vitro site-specific mutagenesis.

6. The Need for chemical synthesis of a nucleotide sequence encoding a specific protein may arise when cloning the corresponding Gene is difficult. In such cases, the gene's nucleotide sequence is deduced from The amino acid sequence of the protein. Chemical synthesis is also employed when the codons comprising a given gene are poorly translated by the host Organism, resulting in very low expression levels. Under these circumstances, a gene can be synthesized with an optimized set of codons (codon optimization) such that the amino acid sequence of the encoded protein remains unchanged, while the codons are translated much more efficiently by the host organism.

Gene Synthesis

If chemically synthesized double-stranded DNA is to be used as a gene or a gene fragment, each of its strands is synthesized separately. Obtaining short genes (60–80 bp) is technically straightforward: complementary strands are synthesized and then annealed. For larger genes (>300 bp), specialized strategies must be employed because the yield of each chemical synthesis cycle never reaches 100%. For example, if a gene consists of 999 base pairs and the stepwise yield is 99%, the proportion of full-length single-stranded DNA at the end of the process will be no more than 0.004%. To overcome this problem, synthetic (double-stranded) genes are assembled from modules—single-stranded fragments ranging from 20 to 100 nucleotides in length.

One approach to constructing a synthetic gene involves generating a set of oligonucleotides, each 20–60 nucleotides long, with overlapping ends. The nucleotide sequences of the strands are designed so that upon annealing, the terminal segments of the gene have blunt ends. Each internal segment features protruding 3' and 5' ends complementary to those of the adjacent segment (Fig. 5.9). Following gene assembly, single-stranded nicks are sealed using T4 DNA ligase. Synthetic genes can be designed to contain, In addition to protein-coding sequences, terminal regions that facilitate insertion into a cloning vector (restriction endonuclease sites) and, if necessary, signal sequences for proper METABOLISM/31.html">Transcription and Translation initiation and termination.

Fig. 5.9. Assembly of a synthetic gene from short oligonucleotides. Individual oligonucleotides, 20 to 60 residues long, are synthesized with nucleotide sequences designed to form a double-stranded molecule upon annealing. Remaining single-stranded nicks are sealed using T4 DNA ligase.

single-stranded nicks using T4 DNA ligase. Synthetic genes can be designed to contain, in addition to protein-coding sequences, terminal regions that facilitate their insertion into a cloning vector (restriction endonuclease sites) as well as, if necessary, signal sequences for proper transcription and translation initiation and termination.

Alternatively, full-length genes can be obtained by first synthesizing a specific set of overlapping oligonucleotides ranging from 40 to 100 residues in length. Upon annealing, 6–10 mutually complementary 3' and 5' terminal nucleotides pair up, leaving large gaps between them. The length of the paired regions is sufficient to stabilize the entire Structure. These gaps are filled enzymatically using Escherichia coli DNA polymerase I, which utilizes 3'-hydroxyl groups to prime replication and single-stranded regions as templates. Remaining single-stranded nicks are sealed with T4 DNA ligase (Fig. 5.10).

Larger genes (>1000 bp) are typically assembled from double-stranded fragments, each consisting of 4–6 overlapping oligonucleotides (20–60 bp each). If sufficient amounts of these fragments are obtained after synthesis and annealing, they are simply joined together. Otherwise, each fragment is cloned and amplified. The double-stranded fragments are sequentially joined until the full-length gene is formed. To verify the accuracy of the nucleotide sequence of the chemically synthesized gene, every double-stranded fragment is sequenced, followed by sequencing of the entire gene.

Fig. 5.10. In vitro assembly of an extended gene involving Enzymes. First, individual oligonucleotides are chemically synthesized with nucleotide sequences designed to form 6–10 base pair paired regions upon annealing. The gaps between them are filled using E. coli DNA polymerase I, and single-stranded nicks are sealed with T4 DNA ligase.