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Sample Submission

AssayDesign PCRSetp Pyrosequencing


Pyrosequencing Design

Assay Design§

  • Ready-Made Assays for pyrosequencing are available from Qiagen.
    • The assays consist of PCR primers and pyrosequencing primers designed for use with PyroMark systems. ~60,000 PyroMark CpG Assays for human, mouse, and rat gene sequences are available in convenient tube or 96-well formats. Custom-designed assays can also be ordered through Qiagen.
  • Custom Assays should be designed using Qiagen's PyroMark Assay Design software.
    • The software will generate PCR and pyrosequencing primers optimized for use with the PyroMark Q24. The software is available for FREE to our customers. To obtain a copy please contact us. We can provide assistance with training and design of pyrosequencing assays.
  • Specific SNP and CpG assay design guidelines can be found within the software's help menu.
  • General guidelines for pyrosequencing assays can be found below.
    • ONE PCR PRIMER MUST BE BIOTIN-LABELED at the 5' end and should be purified.
    • SNP PCR primers should be 18-25bp, with annealing temp of 60-70°C.
    • CpG PCR primers should be 22-30bp.
    • PCR AMPLICON length should be between 50-200bp.
    • TARGET REGION should be less than 100 bases.
    • Pyrosequencing primer should be 15-20 bases long, with annealing temp of 45-55°C.
    • Pyrosequencing primer should be located 1-5 bases away from first SNP or CpG site.
    • Avoid long stretches of homopolyers*, especially next to variable regions (SNP/CpG sites).
    • Avoid strong hairpins and primer dimers.
PCR Setup§


  • DNA concentration should be ~10-20ng/ul per PCR reaction (according to manufacturer's guidelines).
  • PCR primer final concentration should be 0.2uM.
  • Total PCR volume of 20-25ul should be sufficient for pyrosequencing.
  • Run at least 45 cycles of PCR.
  • Load ~1/10th of the PCR product on 1.5% agarose gel. Check for a clear, strong product band (i.e. no excess primer, primer-dimers or other non-specific products**).
  • PCR reaction should not be purified before Pyrosequencing run.




  • Submit ~20ul unpurified PCR product per assay run.
  • Submit primers at 5uM (5pmol/ul) concentration.
  • Provide ~5ul of primer for each rxn.
  • A PyroMark Assay Design file (.xml file) must be email to us.


§ Qiagen recommeded starting conditions. Conditions may need to be adjusted based on reagents used and gel results.
* Avoid homopolymers stretches longer than 5 bases.
** If multiple bands are observed in gel please contact us for assistance.
  • More information about pyrosequencing can be found on our helpful links page
  • Please contact us for submission requirements for LUMA Analysis.