Sequencing-by-Synthesis

More modern and efficient ways of generating DNA sequence information are based on sequencing-by-synthesis. That is, a by-product of an individual nucleotide addition to a growing strand is detected in situ as DNA Pol creates a new strand. For example, the H+ ion (in the case of Ion Torrent) or the inorganic phosphate (in the case of pyrosequencing), that is released upon nucleotide addition may be detected in relatively real time.

Pyrosequencing is one kind of sequencing-by-synthesis. Large DNA templates are fragmented and fixed to the surface of many tiny wells within a microtiter-like dish or chip. These DNA templates are sequenced in parallel, allowing for much more rapid and high-throughput acquisition of sequence information. Enzymes and a single kind of dNTP (A, C, G or T) is added to the wells. If the template strand calls for the dNTP that has been provided, DNA Pol will add it to the new strand. As a result of this chemical linkage, an inorganic pyrophosphate (PPi) is released. The PPi is captured by another enzyme called sulfurylase, the result of which is the production of ATP. ATP is then used by a third enzyme, luciferase,29 to create light. The intensity of emitted light is detected in association with each well of the dish. If a single nucleotide is added to a growing strand, then one unit of light is emitted. If the template calls for the addition of multiple nucleotides of the type provided (e.g., two consecutive Gs), then the light intensity increases proportionally. At the end of the cycle, the apyrase enzyme degrades the unused dNTPs, thus clearing out the system and preparing the reaction for the next round. In the next cycle, a different dNTP is added and the process continues, with each nucleotide being added in a predefined sequential manner. In the end, a pyrogram is generated to describe the light emitted at the end of each cycle of nucleotide addition. The intensity of the light emission can be used to determine the sequence of the newly formed strand.

 
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