Pyrosequencing is a sequencing by synthesis technique that uses a luciferase enzyme system to monitor DNA synthesis. It works by adding DNA polymerase and a single nucleotide to the DNA fragments, generating pyrophosphate that is converted to light. The light is detected and identifies the nucleotide incorporated. Pyrosequencing has applications in cDNA analysis, mutation detection, re-sequencing of disease genes, and identifying single nucleotide polymorphisms and typing bacteria and viruses.

1. Pyrosequencing
By: Qadardana kakar
BS Biotechnology
7th semester

2. Aims/ Objective
 The Aim of presentation is to explain the process of pyrosequencing.

3. Content
 DNA sequencing
 Pyrosequencing
 Principle
 Methodology
 Application of pyrosequencing

4. DNA sequencing
 Technology that is used to determine the order of the four bases adenine, thymine, guanine,
and cytosine in a strand of DNA.
1. Maxam – Gilbert sequencing
 2. Chain-termination methods (Sanger sequencing)
 3. Dye-terminator sequencing
 4. Pyrosequencing (modern techniques)

5. Pyrosequencing
Pyrosequencing technology is a recently established non electrophoretic,
sequencing-by-synthesis technique which uses an enzymatic system based on luciferase to
monitor DNA production .
Principle :
(DNA)n +dXTP DNA polymerase (DNA)n+1 + PPi
PPi+ APS ATP Sufurylase ATP + SO4
2_
ATP + Luciferin+ O2 Luciferase AMP + PPi + Oxyluciferin + CO2 + Light
ATP + dXTP Apyrase ADP+ dXDP + 2Pi
ADP + dXDP Apyrase AMP + dXMP + 2Pi (Wong et al., 1991)

6. Methodology
1. DNA fragmentation
2. Adapter binding to ends of DNA fragments.
3. Denaturation of fragments
4. Fix to a solid surface, Sepharose beads or streptavidin-coated magnetic beads
( beads which are surrounded by single stranded sequences)
5. Beads has sequences complementary to adapter sequences.

7. 1. Methodology
 Load the beads into sequencing wells (small DNA grooves).
 Chemical process
I. Add DNA polymerase and single dNTP (polymerization)
II. Add sulfurylase and APS ( ATP synthesis)
III. Add luciferin and luciferase (light production)
IV. Detection ( light sensor)
V. Washing or add enzyme apyrase enzyme.

9. Application of Pyrosequencing
 Pyrosequencing has shown well performance in determination of complex DNA structure
such as

 cDNA analysis, mutation detection

 Re-sequencing of diseases linked genes,
 viral typing, bacterial typing, and SNPs.