Assay Design

Qiagen Assay Design Software is recommended for pyrosequencing. The software is available for FREE to our customers, to obtain a copy please contact us. We provide assistance with training and design of pyrosequencing assays. An assay design MUST be submitted to the PAN Facility prior to run.

Qiagen Recommendations

• PCR primers should be 18-24 bases long, with anneal temp of 50-68°C.
• One PCR primer MUST BE biotinylated at the 5' end, and should be purified.
• Amplicon lengths should be between 50-300bp.

• Pyro-Sequencing primer must be in the opposite direction of the biotinylated primer
• Pyro-Sequencing primer should be 15-20 bases long, with annealing temp of 45-55°C.
• Pyro-Sequencing run should be less than 100 bases.

• Considerations for CpG Assays
  • PCR primers should be 22-30 bases long, with annealing temp of 56°C.
  • Pyrosequencing primer should be 18-23 bases long.
  • Amplicons should be less than 200bp.

Sample Concentration

• For PCR, sample concentration should be ~50ng/rxn
• For bisulfite treatment, sample concentration should be 500ng-2ug
• DNA should be purified prior to PCR, but does not need to be purified before Pyro-Sequencing run.

Primer Concentration

• Please submit PCR and Pyro-Sequencing primers at a 5uM (5pmol/ul) concentration. Please provide ~5ul of primer for each each rxn.

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