Pyrosequencing

the Pyrosequencing with steps

1. Pyrosequencing By: Qadardana kakar BS Biotechnology 7 th 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  T echnology 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. P yrosequencing (modern techniques)

5.   Pyrosequencing P yrosequencing technology is a recently established non electrophoretic, sequencing-by-synthesis technique which uses an enzymatic system based on luciferase to monitor DNA production . Principle :

6. Methodology DNA fragmentation Adapter binding to ends of DNA fragments. Denaturation of fragments 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. Methodology Load the beads into sequencing wells (small DNA grooves). Chemical process Add DNA polymerase and single dNTP (polymerization) Add sulfurylase and APS ( ATP synthesis) Add luciferin and luciferase (light production) Detection ( light sensor) Washing or add enzyme apyrase enzyme.

8.   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.