Download - Pyrosequencing
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Pyrosequencing
By: Qadardana kakar
BS Biotechnology
7th semester
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Aims/ Objective
The Aim of presentation is to explain the process of pyrosequencing.
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Content
DNA sequencing Pyrosequencing Principle Methodology Application of pyrosequencing
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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)
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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)
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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.
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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.
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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.