Pyrosequencing

By: Qadardana kakar

BS Biotechnology

7th semester

Aims/ Objective

The Aim of presentation is to explain the process of pyrosequencing.

Content

DNA sequencing Pyrosequencing Principle Methodology Application of pyrosequencing

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)

  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 + SO42_

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)

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.

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.

 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.