How do I validate a new Pyrosequencing assay?

All new assays have to be validated by the user. Interaction between primers or loops formed on single-strangled DNA can serve as priming sites for base incorporation by DNA polymerase. The following controls should be included when an assay is analyzed for the first time:

 

 1. PCR without template DNA —shows if the primers interact to give a background signal in Pyrosequencing reactions

 

2. PCR with template DNA but with no sequencing primer — shows if the template can loop back on itself and give a background signal in Pyrosequencing reactions

 

3. Sequencing primer without any PCR product — shows if the sequencing primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

 

4. Biotinylated primer without any PCR product — shows if the biotinylated primer can form duplexes or hairpins and give background signal in Pyrosequencing reactions

 

5. Sequencing primer and biotinylated primer together without PCR product —shows if the sequencing primer and the biotinylated primer can form duplexes and give background signal in Pyrosequencing reactions

 

Programs from these controls ought not to show any significant peaks after any nucleotide addition.