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 of sequences of 300 to 500 nucleotides per run.
Sequencing Using Reversible Chain Terminators
For pyrosequencing, each of the four nucleotides must be added sequentially in separate cycles. The sequence of a DNA fragment could be determined more rapidly if all the nucleotides were added together in each cycle. However, the reaction must be controlled to ensure that only a single nucleotide is incorporated during each cycle, and it must be possible to distinguish each of the four nucleotides. Synthetic nucleotides known as reversible chain terminators have been designed to meet these criteria and form the basis of some of the next-generation sequencing-by-synthesis technologies.
Reversible chain terminators are deoxynucleoside triphosphates with two important modifications: (i) a chemical blocking group is added to the 3′ carbon of the sugar moiety to prevent addition of more than one nucleotide during each round of sequencing and (ii) a different fluorescent dye is added to each of the four nucleotides to enable identification of the incorporated nucleotide (Fig. 2.37A). The fluorophore is added at a position that does not interfere with either base-pairing or phosphodiester bond formation. Similar to the case with other sequencing-by-synthesis methods, DNA polymerase is employed to catalyze the addition of the modified nucleotides to an oligonucleotide primer as specified by the DNA template sequence (Fig. 2.37B). After recording fluorescent emissions, the fluorescent dye and the 3′ blocking group are removed. The blocking group is removed in a manner that restores the 3′ hydroxyl group of the sugar to enable subsequent addition of another nucleotide in the next cycle. Cycles of nucleotide addition to the growing DNA strand by DNA polymerase, acquisition of fluorescence data, and chemical cleavage of the blocking and dye groups are repeated to generate short read lengths (i.e., 50 to 100 nucleotides per run).
Figure 2.37 Sequencing using reversible chain terminators. (A) Reversible chain terminators are modified nucleotides that have a removable blocking group on the oxygen of the 3′ position of the deoxyribose sugar to prevent addition of more than one nucleotide per sequencing cycle. To enable identification, a different fluorescent dye is attached to each of the four nucleotides via a cleavable linker. Shown is the fluorescent dye attached to adenine. (B) An adaptor sequence is added to the 3′ end of the DNA sequencing template that provides a binding site for a sequencing primer. All four modified nucleotides are added in a single cycle, and a modified DNA polymerase extends the growing DNA chain by one nucleotide per cycle. Fluorescence is detected, and then the dye and the 3′ blocking group are cleaved before the next cycle. Removal of the blocking group restores the 3′ hydroxyl group for addition of the next nucleotide.
Sequencing by Single Molecule Synthesis
To generate sufficiently high levels of a fluorescent or light signal for detection of nucleotide addition, the sequencing methods described above require large amounts of template DNA. A DNA amplification step is often required, which increases template preparation time and can introduce mutations that are interpreted as nucleotide variations. Recently, sequencing technologies have been developed to circumvent the amplification step. In one approach, a single molecule of DNA polymerase is immobilized on a solid support (on the bottom of a nanoscale well) and captures a single DNA molecule that is bound to a primer (Fig. 2.38A). During the sequence acquisition stage, DNA polymerase extends the primer in a template dependent fashion and a signal corresponding to nucleotide addition is measured in a narrow volume at the bottom of the well (Fig. 2.38B).