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Overview of Pyrosequencing-Short Read on PSQ96 (THIS IS NOT NEXTGEN SEQUENCING)

The UCLA Sequencing Core in the Department of Human Genetics provides a PSQ96 Pyrosequencer. PYROSEQUENCING technology is a unique method for short-read DNA sequencing,Allele Quantification and mutation/SNP analysis. Its ease of use, sequence validation and flexibility make it ideally suited for applied genomics research including molecular applications for disease diagnosis, clinical prognosis and pharmacogenomics testing. Typical run times are 10 minutes for 96 samples and approximately 30 to 45 minutes for sequence analysis applications that routinely provide 30 to 50 bases of sequence information. No gels or dye labels are needed.

Click here for the pyrosequencing protocol.

You may visit www.pyrosequencing.com and techsupport.pyrosequencing.com for in depth information.


Principle of Pyrosequencing

Pyrosequencing™ is sequencing by synthesis, a simple to use technique for accurate and consistent analysis of DNA sequences.

Step 1

A sequencing primer is hybridized to a single stranded, PCR amplified, DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin.

Step 2

The first of four deoxynucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.

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Step 3

ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Each light signal is proportional to the number of nucleotides incorporated.

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Step 4

Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.

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Step 5

Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase.

As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the pyrogram.

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Prices

Ready to run Pyrosequencing - $220.00 (one 96 well plate, regardless of number of samples) Primer design- please contact us for a quote


For more information please contact:

Hemani Wijesuriya
Email: HWijesuriya@mednet.ucla.edu
Phone: 310-267-2461
Uma Dandekar, GenoSeq Core Assistant Director
Email: uma@ucla.edu
Phone: 310-267-2461
Fax: 310-794-5446
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