PYROSEQUENCING
Introduction
Principle
Working
Oil emulsion PCR:
(image2. Oil Emulsion PCR) |
Isolate the pure gDNA and bind it with biotin as more adept. Now, hybridize it with streptavidin bounded bead. Add NaOH to denature the DNA fragment. add this mixture into the centrifuge tube and centrifuge it, in which DNA fragments bound with streptavidin bead are remain in the pellet and the complementary strand are present into the supernatant. After the washing step, the complementary strand is discarded. The DNA fragment bound with streptavidin bead, now add in the oil-emulsion PCR plate and set up it into oil-emulsion PCR machine. In this PCR reaction, a small water droplet acts as a small individual PCR system. After the amplification, the bead contains many copies of the same DNA fragment.
(image3. DNA polymerase adds dNTP in the growing chain and releases PPi that can utilize in further reaction and at the end of the reaction light will be produced.) |
- In the cheap, each bead is fixed into each well. Excessive beads are removed by washing.
- Now, sequencing primer bind with DNA strand on the streptavidin bead, and then DNA polymerase comes and binds with DNA-primer hybrid and initiates the process.
- When the appropriate nucleotide comes into the sequencing system, the DNA polymerase attaches it on 3'-end of the growing chain and release pyrophosphate (PPi).
- Free pyrophosphate and APS (Adenosine 5'- phosphosulfate) come in contact with an enzyme called APS sulfurase, which is converted APS into ATP and release sulfate ions.
- Now ATP comes in contact with Luciferase which is convert luciferin into oxyluciferin and produces light. This light can be detected by the machine which is after converted into the signal and the computer can convert this signal into an appropriate result.
- The intensity of the light produced by different nucleotides is different and also depends on the number of nucleotides.
- Now the enzyme named Apyrase degrades the excessive ATP as well as an unused nucleotide.
Detection
(image4. program. in this graph picks indicate the intensity of light which is dependent on nucleotide and the number of nucleotides.) |
Pros
- It gives high throughput (> 200,000)
- RaRaRapidAPIhod with real-time readout. That is highly reliable for sequencing short strands of DNA.
- It can generate a sequence signal immediately downstream of the primer.
- It gives simple frequency data.
Cons
- very expensive method.
- this method has a relatively high error rate.
- Long single nucleotide sequence not reliable.
- The long fusion of primers may bring bias.
Thank you for this post.its very usefull .
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