Pyrosequencing | Introduction, Principle, Working method, Pros and Cons

PYROSEQUENCING

By Meet Patel

Introduction

Pyrosequencing was firstly described in 1993 by Bertil Pettersson, Mathias then, and Pål Nyren combined the solid phase sequencing method using streptavidin (coated with magnetic beads) with DNA polymerase lacking proofreading activity and luminescence detection by the luciferase enzyme. The second method describes in 1998 by Mostafa Ronaghi, uhlen, and Pål Nyren, they include additional enzyme apyrase which is removed nucleotide that is not incorporated by the DNA polymerase. Pyros means in Greek is “Fire”. A Swedish company known as PYROSEQUENCING marked this method for short fragments of DNA. This method can read approximately 200-300 base lengths and is able to sequence 400,000 reactions at a time. 

Principle

In this method of DNA sequencing, a dNTP is attached to the 3’- end of the growing chain and released two phosphate molecules as pyrophosphate (PPi) (That is why the method is called pyrosequencing). ATP sulfurylase use this PPi and APS (Adenosine 5’ phosphosulfate) to produce ATP. Luciferase and ATP as substrate are converting luciferin into oxyluciferin and released light. The amount of light is proportional to the number of ATP. After the reaction is completed, Apyrase is destroyed by the excessive non-usable dNTPs.

  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.

Sequencing:

(image3. sequencing microtiter well. Each well contains s single bead.)

Now DNA fragments pipette onto the sequencing cheap (microtiter cheap). This cheap, now, put into the sequencing machine. The machine automatically adds the following reagents; Sequencing Primer, APS, Luciferin, DNA polymerase, ATP sulfurylase, luciferase, and apyrase.

(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.)

The light produced during sequencing is dictated by a CCD camera which produces an electric signal and sends a signal to the computer. A computer converts this electric signal ina aa an an an an a to graph known as "PROGRAM.

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.