Pyrosequencing(R) A Simple Method for Accurate Genotyping part 1

Pyrosequencing(R) A Simple Method for Accurate Genotyping part 1

  Pyrosequencing is a synthesis based DNA sequencing method developed by Mostafa Ronaghi and Pal Nyren in 1996 (1). The sequence of a single stranded DNA fragment is determined by synthesizing its complementary strand and monitoring incorporation of the nucleotide at each base pair position. Single nucleotide solutions (A/T/C/G) are sequentially applied and then removed. When the proper nucleotide is added, pyrophosphate (PPi) is released. ATP sulfurylase converts PPi to ATP which is then used as an energy source for luciferase to convert luciferin to oxyluciferin, producing light (2). A camera detects the light emission and software is used to analyze and compile results.Write the first paragraph of your page here.

Array Based PyrosequencingEdit

Pyrosequencing techniques, like many genetic tools, have been modified 


to accommodate the ever increasing amount of genomic/metagenomic data. 454 Life Sciences developed an array based pyrosequencing technology that is more adept at handling genomic/metagenomic sized data sets (1). The tehcnology utilizes magnetic beads housed in wells to bind short (400-500 bp) DNA templates. Droplets containing enzymes and deoxyribonucleotide triphosphates are applied to the beads in sequential order, and correct base pair matches will result in the emission of light as in the earlier versions of pyrosequencing. Beads can then be washed thoroughly, removing excess dNTPs, without stripping the template due to the high affinity caused by the magnetism. By utilizing multiple well arrays with large bead sets, sequ

encing efficiency is greatly increased. In one ten hour run 400 MB of sequence data can be generated  (3. Array based pyrosequencing offers a lost cost option to scientists working with large data sets.



2. A Sequencing Method Based on Real-Time Pyrophosphate Mostafa Ronaghi, Mathias Uhlen, and Pal Nyren. Science 17 July 1998: 281 (5375), 363-365. [DOI:10. 1126/science281.5375.363.]

3. Droplet-Based Pyrosequencing Using Digital Microfluidics. Deborah J. Boles, Jonathan L. Benton, Germaine J. Siew, Miriam H. Levy, Prasanna K. Thwar, Melissa A. Sandahl, Jeremy L. Rouse, Lisa C. Perkins, Arjun P. Sudarsan, Roxana Jalili, Vamsee K. Pamula, Vijay Srinivasan, Richard B. Fair, Peter B. Griffin, Allen E. Eckhardt, and Michael G. Pollak. Analytical Chemistry 2011 83 (22), 8439-8447