454 Sequencing

In 2005, new sequence technique, which known as next-generation or second generation sequencing, was emerged after three decades of Sanger sequencing dominance. The new technique, 454 pyrosequencing, has completely new mechanism compared with the previous one, where this technique depended on series of enzymatic steps that including four enzymes polymerase, ATP sulfurylase, luciferase and apyrase under pyrosequencing mechanism. The name 454 pyrosequencing derived from pyrophosphate that realised from incorporation of nucleotide to new DNA strand during sequencing run. Four main steps summarises the entire sequencing technology, that have described in 454 sequencing website, (i) DNA library preparation, (ii) Emulsion PCR, (iii) Sequencing-by-synthesis …show more content…
In emulsion, a water micro-reactors are forming around the beads that have only one library fragment (1:1 bead to fragment ratio) with a combination of amplification materials. Essentially, once the PCR process is completed, the result of this process is screened and only the amplified and enriched beads are kept while others that are not carrying any amplified DNA are eliminated. Following the enriched beads filtration, distribution of these beads to the PicoTiterPlate will be performed, mathematically, the dimensions of the PicoTiterPlate wells and beads, 44 and 28 μm in diameter respectively, are designed to allow only one bead fit per well. At the same time, smaller beads, bearing immobilized pyrosequencing enzymes (ATP sulfurylase, luciferase and apyrase), are incubated into wells with enriched beads, polymerase, single-stranded binding protein, while luciferin, oxygen and other chemical reagents required for sequencing by synthesis reaction are supplied through a …show more content…
Briefly, when polymerase adds nucleotides to the bead-captured primers depending on the bases of DNA template strands, the pyrophosphate (PPi) will be released, then ATP sulphurylase together with present of adenosine phosphosulphate (APS) will catalyze PPi and form adenosine triphosphate (ATP). Another enzyme, luciferase, is used the preformed ATP as an energy to transfer luciferin, that added with oxygen, to oxyluciferin and generate visible light. The amount of emitted light equals the number of incorporated dNTPs. Finally, to stop the emission reaction the enzyme apyrase will be used, this enzyme degrades ATP and it has another mission which is that degradation of the unincorporated nucleotides. Consequently, the environment will be cleaned from unmatched bases that can produce a high levels of noise and confusing result if they still without removing in the next cycle of adding new dNTP because, for this process, each type of dNTPs are added separately at each cycle of pyrosequencing