Next Generation Sequencing Technologies

NEXT GENERATION SEQUENCING TECHNOLOGIES Name Institution Affiliation Course Date of Submission Next Generation Technologies Article selected - Pyrosequencing: Principles and Applications 1. How the technology works. Pyrosequencing is sequency by synthesis technology. To begin with hybridization is done to sequencing primer. The primer will then become a single- stranded PCR amplicon which will act as a template. The template will then be incubated with different enzymes which include apyrase, ATP sulfurylase ,DNA polymerase, , luciferase,. Substrates like luciferin and adenosine 5' phosphosulfate (APS) will also be used. The second step involves addition of deoxribonucleotide triphosphate (dNTP) to the reaction at hand. IN the process, there is catalysation of the deoxyribo-nucleotide triphosphate into the DNA strand (Marsh, 2007). This is done by the DNA polymerase enzyme. Note that this only takes p[lace if the two are complementary. In the event that they are complementary, pyrophosphate (PPi) is released. The quantity released is always dependent to the nucleotide that is used. PPi is then coinverted to ATP by ATP sulfurylase enzyme (Rapley et al., 2012). This only happens when adenosine 5' phosphosulfate (APS) is in place. luciferase-catalyzed reaction then occurs when the ATP changes the conversion of luciferase to oxyluciferin. This process lead to the generation of light proportionally to ATP amounts used. Detection of the light is only by the device that is charged. It is visible in the Pyrogram. The nucleotides and ATP are degraded by the apyrase. A new nucleotide is put in when the degradation ends (Fakruddin et al., 2008). The deoxyadenosine triphosphate (dATP) can be substituted by deoxyadenosine alfa-thio triphosphate (dATP·S) in the process. Moreover, the complementary DNA is built up as the entire process is in progress. The signal peaks in the trace of Pyrogram influences the nucleotide sequence. 2. How it is applied to solve a particular problem - This technology can be used in clinical genetic applications. Pyrosequencing technology is used to detec...