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 The Sanger method superseded the Maxam and Gilbert sequencing procedure when the M13 bacteriophage cloning system was developed, which provided single-stranded DNA templates required for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 are represented as a sequencing chromatogram (colored peaks) (C).
PCR-based cycle sequencing is performed to minimize the amount of template DNA required for sequencing. Multiple cycles of denaturation, primer annealing, and primer extension produce large amounts of dideoxynucleotide-terminated fragments. These are applied to a polymer in a long capillary tube that enables separation of DNA fragments that differ in size by a single nucleotide. As each successive fluorescently labeled fragment moves through the polymeric matrix in an electric field and passes by a laser, the fluorescent dye is excited. Each of the four different fluorescent dyes emits a characteristic wavelength of light that represents a particular nucleotide, and the order of the fluorescent signals corresponds to the sequence of nucleotides (Fig. 2.35C). Generally, automated systems that employ this sequencing technology can determine with high accuracy about 500 to 600 bases per run (the read length, or read).
Pyrosequencing
Pyrosequencing was the first of the next-generation sequencing technologies to be made commercially available. The basis of the technique is the detection of pyrophosphate that is released during DNA synthesis. When a DNA strand is extended by DNA polymerase, the α-phosphate attached to the 5′ carbon of the sugar of an incoming deoxynucleoside triphosphate forms a phosphodiester bond with the 3′ hydroxyl group of the last nucleotide of the growing strand. The terminal β- and γ-phosphates of the added nucleotide are cleaved off as a unit known as pyrophosphate (Fig. 2.36A). The release of pyrophosphate correlates with the incorporation of a specific nucleotide in the growing DNA strand.
Figure 2.36 Pyrosequencing is based on the detection of pyrophosphate that is released during DNA synthesis. (A) A phosphodiester bond forms between the 3′ hydroxyl group of the deoxyribose sugar of the last incorporated nucleotide and the α-phosphate of the incoming nucleotide (blue arrow). The bond between the α- and β-phosphates is cleaved (green arrow), and pyrophosphate is released (black arrow). (B) An adaptor sequence is added to the 3′ end of the DNA sequencing template that provides a binding site for a sequencing primer. One nucleotide (deoxyribonucleoside triphosphate [dNTP]) is added at a time. If the dNTP is added by DNA polymerase to the end of the growing DNA strand, pyrophosphate (PPi) is released and detected indirectly by the synthesis of ATP. ATP is required for light generation by luciferase. The DNA sequence is determined by correlating light emission with incorporation of a particular dNTP.
To determine the sequence of a DNA fragment by pyrosequencing, a short DNA adaptor that serves as a binding site for a sequencing primer is first added to the end of the DNA template (Fig. 2.36B). Following annealing of the sequencing primer to the complementary adaptor sequence, one deoxynucleotide is introduced at a time in the presence of DNA polymerase. Pyrophosphate is released only when the complementary nucleotide is incorporated at the end of the growing strand. Nucleotides that are not complementary to the template strand are not incorporated, and no pyrophosphate is formed.
The pyrophosphate released following incorporation of a nucleotide is detected indirectly after enzymatic synthesis of ATP (Fig. 2.36B). Pyrophosphate combines with adenosine-5′-phosphosulfate in the presence of the enzyme ATP sulfurylase to form ATP. In turn, ATP drives the conversion of luciferin to oxyluciferin by the enzyme luciferase, a reaction that generates light. Detection of light after each cycle of nucleotide addition and enzymatic reactions indicates the incorporation of a complementary nucleotide. The amount of light generated after the addition of a particular nucleotide is proportional to the number of nucleotides that are incorporated in the growing strand, and therefore sequences containing tracts of up to eight identical nucleotides in a row can be determined. Because the natural nucleotide dATP can participate in the luciferase reaction, dATP is replaced with deoxyadenosine α-thiotriphosphate, which can be incorporated into the growing DNA strand by DNA polymerase but is not a substrate for luciferase. Repeated cycles of nucleotide addition, pyrophosphate release, and light detection enable determination
