PyroMark Sequencing

Contact: Cassie Sather
Location: Thomas Bldg DE-740
Contact phone: (206) 667-2757
Contact fax: (206) 667-2825
Contact e-mail: csather@fredhutch.org

The PyroMark Q24 and PyroMark Q96 MD are sequencing-by-synthesis platforms that use pyrosequencing for analysis of short to medium length DNA sequences.  The PyroMark is used to study CpG methylation, allele quantification and mutation analysis in real-time and can accommodate 24 samples (Q24 platform) or 96 samples (Q96 MD platform) in parallel.

Sequencing-by-Synthesis

Samples are prepared in parallel from PCR product to single stranded template ready for sequencing.  Through a reaction cascade system, the single stranded template is used as one nucleotide is incorporated at a time, generating pyrophosphate.  Pyrophosphate is then converted into ATP which drives the luciferase-mediated conversion of luciferin to oxyluciferin.  The result is visible light that is generated 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) chip and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated.  Apyrase is added to degrade all unincorporated nucleotides and followed by the addition of the next nucleotide.

Please refer to this website for more information on the principle of pyrosequencing.

How to Get Started

Proper assay and primer design are critical for a successful Pyrosequencing project.  A consultation with a member of the Genomics department is required prior to submitting a new project.  To request more information or schedule a consultation, please contact Cassie Sather (email: csather@fredhutch.org; phone (206) 667-2757) or Crissa Bennett (email: ckbennet@fredhutch.org; phone (206) 667-5693).