Mysterious peaks in pyrosequencing runs!?pyrosequencing
Posted 06 August 2012 - 04:07 AM
Our lab recently started to use pyrosequencing for quantitative methylation analysis. We all do not have a lot experience and expertise with this technique.
I am currently investigating the methylation status of one gene in tumor tissue samples. I have sequenced more than hundred samples and for a few samples I made the following observation: The pyrogram shows peaks at dispensations, where no peaks should occur. When I analyze the corresponding PCR products by gel electroforesis, I see a nice band at the good size, plus a slight band below the good band. It looks like a dimer, but why do I only see this in combination with the peaks? Could this also be contamination?
I always include a negative control (no DNA in PCR mix) in the pyrosequencing runs, which do not give any results (= clean). I checked my primers with MethylBlast and they should be specific.
I would be more than happy, if anyone could help me looking for a reason for those peaks/bands!! Did someone make similar observations?
I cannot analyze my data until I know, what I am dealing with...
Posted 07 August 2012 - 02:21 AM
Posted 13 August 2012 - 03:51 AM