Molecular Biotechnology. Bernard R. Glick

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 of nucleotides. This approach was time-consuming and tedious. With the advent of the dideoxy method, it became obsolete. The Sanger method superseded the Maxam and Gilbert sequencing procedure when the M13 bacteriophage cloning system was developed, which provided single-stranded DNA templates required for sequencing. The M13 system was no longer required following introduction of PCR-based cycle sequencing, which generates single-stranded DNA templates during a DNA denaturation step. Sanger and Gilbert received the Nobel Prize in Chemistry in 1980 for their work.

Dideoxynucleotide Procedure

      The dideoxynucleotide procedure for DNA sequencing is based on the principle that during DNA synthesis, addition of a nucleotide triphosphate requires a free hydroxyl group on the 3′ carbon of the sugar of the last nucleotide of the growing DNA strand (Fig. 2.34A). However, if a synthetic dideoxynucleotide that lacks a hydroxyl group at the 3′ carbon of the sugar moiety is incorporated at the end of the growing chain, DNA synthesis stops because a phosphodiester bond cannot be formed with the next incoming nucleotide (Fig. 2.34B). The termination of DNA synthesis is the defining feature of the dideoxynucleotide DNA sequencing method.

      Figure 2.34 Incorporation of a dideoxynucleotide terminates DNA synthesis. (A) Addition of an incoming deoxyribonucleoside triphosphate (dNTP) requires a hydroxyl group on the 3′ carbon of the last nucleotide of a growing DNA strand. (B) DNA synthesis stops if a synthetic dideoxyribonucleotide that lacks a 3′ hydroxyl group is incorporated at the end of the growing chain because a phosphodiester bond cannot be formed with the next incoming nucleotide.

      In a dideoxynucleotide DNA sequencing procedure, a synthetic oligonucleotide primer (∼17 to 24 nucleotides) anneals to a predetermined site on the strand of the DNA to be sequenced (Fig. 2.35A). The oligonucleotide primer defines the beginning of the region to be sequenced and provides a 3′ hydroxyl group for the initiation of DNA synthesis. The reaction tube contains a mixture of the four deoxyribonucleotides (deoxyadenosine triphosphate [dATP], deoxycytidine triphosphate [dCTP], deoxyguanosine triphosphate [dGTP], and deoxythymidine triphosphate [dTTP]) and four dideoxynucleotides (dideoxyadenosine triphosphate [ddATP], ddCTP, ddGTP, and ddTTP). Each dideoxynucleotide is labeled with a different fluorescent dye. The concentration of the dideoxynucleotides is optimized to ensure that during DNA synthesis a modified DNA polymerase incorporates a dideoxynucleotide into the mixture of growing DNA strands at every possible position. Thus, the products of the reaction are DNA molecules of all possible lengths, each of which includes the primer sequence at its 5′ end and a fluorescently labeled dideoxynucleotide at the 3′ terminus (Fig. 2.35B).

      Figure 2.35 Dideoxynucleotide method for DNA sequencing. An oligonucleotide primer binds to a complementary sequence adjacent to the region to be sequenced in a single-stranded DNA template (A). As DNA synthesis proceeds from the primer, dideoxynucleotides are randomly added to the growing DNA strands, thereby terminating strand extension. This results in DNA molecules of all possible lengths that have a fluorescently labeled dideoxynucleotide at the 3′ end (B). DNA molecules of different sizes are separated by capillary electrophoresis, and as each molecule passes by a laser, a fluorescent signal that corresponds with one of the four dideoxynucleotides is recorded. The successive fluorescent signals