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 (E and F) that overlap the ends of the second-cycle PCR product are added to a sample, and a third PCR cycle is initiated. The final PCR product is a double-stranded DNA molecule with a specified sequence of nucleotides. The pairs of letters with or without a prime (e.g., A′ and A) represent complementary oligonucleotides. Each oligonucleotide corresponds to a sequence from a particular DNA strand.
Thereafter, pairs of oligonucleotides are added, one of the pair overlapping the upstream sequence of the DNA molecule formed in the previous round and the other overlapping the downstream sequence, and subjected to 20 PCR cycles for each pair added until the entire gene is formed. Synthesis of a gene with 1,000 bp can be carried out in one day. As with other methods for assembling genes, the last pair of oligonucleotides (i.e., the 5′ and 3′ ends of the gene) can be made with supplementary sequences outside the coding region that facilitate the cloning of the gene into a vector and, at the 5′ end, with sequences that enable the gene to be expressed in a host cell.
DNA Sequencing Technologies
Determination of the nucleotide composition and order in a gene or genome is a foundational technique in molecular biotechnology. Cloned or PCR-amplified genes and entire genomes are routinely sequenced. DNA sequences can often reveal something about the function of the protein encoded in a gene, for example, from predicted cofactor binding sites, transmembrane domains, receptor recognition sites, or DNA-binding regions. The nucleotide sequences in noncoding regions that do not encode a protein or RNA molecule may provide information about the regulation of a gene. Comparison of gene sequences among individuals can reveal mutations that contribute to phenotypic differences. For example, identification of nucleotide differences (polymorphisms) in a gene in individuals with a particular disease, but not in healthy individuals, may be used to predict disease susceptibility. Comparison of gene sequences among different organisms can lead to the development of hypotheses about the evolutionary relationships among organisms.
For more than three decades, the dideoxynucleotide procedure developed by the English biochemist Frederick Sanger (see Milestone box on page 53) has been used for DNA sequencing. This includes sequencing of DNA fragments containing one to a few genes and also the entire genomes from many different organisms, including the human genome. However, the interest in sequencing large numbers of DNA molecules in less time and at a lower cost has driven the recent development of new sequencing technologies that can process thousands to millions of sequences concurrently. Many different sequencing technologies have been developed. In general, all of these methods involve (i) enzymatic addition of nucleotides to a primer based on complementarity to a template DNA fragment and (ii) detection and identification of the nucleotide(s) added. Most employ DNA polymerase to catalyze the addition of single nucleotides (sequencing by synthesis), although ligase may also be used to add a short, complementary oligonucleotide (sequencing by ligation). The techniques differ in the method by which the addition is detected.
milestone DNA Sequencing with Chain-Terminating Inhibitors
New techniques are the lifeblood of science. They enable researchers to acquire information that was previously inaccessible and that, in turn, generates insights that stimulate new research and lead to new discoveries. For molecular biotechnology, DNA sequencing is a powerful procedure that has become a laboratory mainstay. The most definitive form of molecular characterization of a gene or genome is its sequence. Among other things, the coding content of a gene, potential primer sequences for a PCR, and the presence of mutations can be determined by DNA sequencing.
Sequencing by enzymatic DNA synthesis with chain elongation inhibitors is a relatively simple, accurate, and reliable method developed by Sanger et al. (Proc. Natl. Acad. USA. 74:5463–5467, 1977). At the time the Sanger (dideoxy) method was published, most DNA sequencing was carried out by the base-specific chemical cleavage method devised by A. M. Maxam and W. Gilbert (Proc Natl Acad Sci USA. 74:560–564, 1977). Before the development of these techniques, nucleic acid sequencing was more or less limited to RNA molecules. The sequencing of a DNA molecule required transcribing a DNA fragment into RNA with RNA polymerase and then sequencing the RNA product. In general, RNA sequencing entailed treating a radiolabeled RNA molecule with different ribonucleases, chromatographically separating the digestion products, redigesting the separated products, hydrolyzing the products of the second digestion with alkali, chromatographically separating the hydrolysis products, determining the sequence of the oligonucleotides, and constructing the sequence based on overlapping stretches
