Pyrosequencing is a DNA sequencing method that detects light emitted from the release of pyrophosphate during DNA strand synthesis. It works by sequentially adding nucleotides and detecting if they are incorporated into the new DNA strand based on pyrophosphate emission. The sequence is determined by detecting which nucleotides are incorporated at each step as complementary DNA is synthesized.

1. Pyrosequencing

2. What is Pyrosequencing?
Pyrosequencing is a method of DNA
sequencing that detects light emitted during the
sequential addition of nucleotides during the
synthesis of a complementary strand of DNA.

3. Concept 1: During DNA Strand Synthesis

4. Concept 2: Detection of released pyrophosphatev

5. The pyrophosphate released during DNA synthesis can be
detected by the emission of light. This detection of
pyrophosphate is the basics of DNA sequencing, hence named as
pyrosequencing.

6. Essential Requirements:
1.
2.
3.
4.

7. 5.
6.

8. Steps involved in Pyrosequencing:
Step 1: Incubation of template strand with primer
Step 2: Addition of enzymes and substrates

9. Step 3: Addition of one type of nucleotide
If the added dNTP is incorporated in the new strand

10. If the added dNTP is NOT incorporated in the new strand
Step 4: Enzyme Apyrase is added that removes unused nucleotides

11. Step 5: Reaction starts again with another nucleotide
Reaction Continues

12. Example:
Not incorporated
C Incorporated

13. Remaining sequence continues like that
The amount of light generated is proportional to
the no of nucleotides that are incorporated.

14. Graph prediction:

15. Overview of Pyrosequencing