Pyrosequencing - Principle and Steps

  Pyrosequencing is a DNA sequencing method. In which incorporation of dNTPs in the DNA is detected in a form of life.

Basic principle 

  •  A new DNA strand is synthesized on the template strand.
  • During DNA synthesis a phosphodiester bond is formed between the last nucleotide of the growing strand and the incoming complementary nucleotide. As a result Pyrophosphate (PPi) is released.
  • Thus every time a complementary nucleotide is incorporated in a strand being synthesized, a pyrophosphate will be released.
  • This pyrophosphate is detected in pyrosequencing and forms the basis for the determination of the DNA sequence of the template strand.

Detection of released Pyrophosphate

  The  pyrophosphate is detected by an enzyme cascade reaction that results in the emission of light.
  The emission of light confirms that a pyrophosphate has been released. 

Reactions 1

  When released pyrophosphate combines with substrate known as Adenosine Phosphosulphate (APS) in the presence of an enzyme ATP sulfurylase, The ATP  is generated.
  • PPi + APS ➞ ATP + Sulfate (catalyzed by ATP-sulfurylase)

Reaction 2

In the next reaction this ATP is utilized by the enzyme luciferase for the conversion of Lucifer into Oxyluciferin and production of light.
  • ATP + luciferin + O₂ ➞ AMP + PPi + oxyluciferin + CO₂ + Light (catalyzed by luciferase);
  Thus the pyrophosphate released during DNA synthesis can be detected by the emission of light. This detection of pyrophosphate is the basis of DNA sequencing and hence the name pyrosequencing.

Requirements for pyrosequencing

  • A DNA fragment, That we want a sequence. This DNA fragment will be our template strand. This DNA fragment is engineered at one end with a sequence that is complementary to a primer. 
  • Primer
  • Deoxynucleotides dNTPs (dATPαS, dCTP, dGTP, dTTP), here the normal dATP is replaced by dATPαS. That is deoxyadenosine alpha Theotriphosphate.
  • This replacement is necessary, this is because enzyme luciferase also uses ATP to produce light. To avoid false signals of pyrophosphate protection during pyrosequencing this replacement is done. dATPαS is used by DNA polymerase but not by luciferase.
  • Enzyme DNA polymerase, for new strand synthesis.
  • Other substrates required - Adenosine Phosphosulphate (APS), Luciferin
  • Other enzymes required - ATP sulfurylase, Luciferase, An enzyme known as Apyrase also needed. (This enzyme removes unused nucleotides. Thus it is a nucleotide degrading enzyme.)

Steps involved in Pyrosequencing

Step 1 - Incubation of template strand with the primer

  •    The DNA fragment of unknown sequence is taken. This DNA sequence is engineered at one end that is complementary to a known primer.
  •   This DNA fragment serves as DNA template strand and it is incubated with the primer. The primer binds to its complementary sequence on the DNA template strand.

Step 2 - Addition of Enzymes and Substrate

  • In this step DNA polymerase is added, along with the other enzymes (ATP sulfurylase, Luciferase) and substrates (Adenosine Phosphosulphate, Luciferin) required for the detection of pyrophosphate.

Step 3 - Addition of One Type of Nucleotide

  •  After the second step one of the four types of nucleotides is added.
  • Here note that only one type of nucleotide is added at a time. That means if we are adding dCTP, then the solution of nucleotide we are adding contains only dCTP molecules.
  •  If the added nucleotide is incorporated in the new strand, pyrophosphate will be released and emission of light will take place. This light is detected by a detector and later used to interpret the unknown sequence the details of this we will see shortly.
  • But if the added nucleotide is Not incorporated, then there will be no pyrophosphate and therefore no light emission. This happens when the incoming nucleotide is not complementary to the nucleotide of the template strand.

Step 4 - Enzymes Apyrase is added for removes unused nucleotides

  • Now in both the cases whether the nucleotide is incorporated or not. Extra or unused nucleotide will be present.
  • These extra nucleotides are now removed from the reaction. This is done by adding enzyme Apyrase the nucleotide degrading enzyme.

Step 5 - Reaction starts again with another nucleotide

  •  After the degradation of unused and extra nucleotides is completed. The pyrosequencing reaction starts again with the addition of next nucleotide.
  •  This process is repeated adding each nucleotide one after the other until the synthesis is complete. So every time a complementary nucleotide is added emission of light will take place.

Graphical Presentation

  The light emission in pyrosequencing is represented graphically to interpret the sequence. The Y-axis of graph represents the light intensity and the X-axis represents the sequence in which nucleotides are added.

Graphical presentation of pyrosequencing
 
  The peaks in the graph also give an idea about the number of same nucleotides present in the sequence. So as per the figure sequence of the Strand that is synthesized during pyrosequencing is C D A C D A G G G A. 
The sequence of the template strand will be complementary to this. Thus the sequence of the unknown DNA fragment is found using pyrosequencing.