2. The process of 454 Pyrosequencing: one of Next generation sequencing methods

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The process of 454 Pyrosequencing: one of Next generation sequencing methods

Daeyeol Ye

- Typically, 4 stages in 454 pyrosuencing

Preparing DNA library -> Emulsion PCR -> Loading DNA beads -> Pyrosequencing

a. preparing DNA library

- ligating two adaptors to the gene of interest

* One adaptor is for attaching the gene to the DNA catching bead.

* The other adaptor is for the primer during emulsion PCR.

- After that, denature the gene into two single strands.

 

b. Emulsion PCR

- Before PCR, attach the strand to the DNA capture beads which contains one of the adaptors. And emulsify the beads with PCR reagent in a water-in-oil mixture to form a microreactor for PCR.

- Running PCR to create millions of copies of each strands in the microreactor

 

c. Loading DNA beads

- After PCR, load the beads onto the PicoTiterPlate device, where the surface design allow for only one bead per well. The PTP Device is then loaded in instrument for sequencing.

- Individual nucleotides are flowed in sequence across the wells. Each incorporation of a nucleotide complementary to the template strand results in a chemiluminescent light signal recorded by the camera.

 

d. Pyrosequencing

- Before pyrosequencing, put the enzyme bead including sulfurylase and luciferase

- During replication, a single pyrophosphate molecule comes out when adding a single nucleotide.

- Then ATP molecule is made by sulfurylase. And then, energy in the ATP is converted into the light energy by luciferase. By measuring it, the sequence of DNA can be determined.