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EPIGENETICS-NEWBIE-Weird/too close primers for pyrosequencing assay - (Feb/29/2012 )

Dear Forum experts,

I am very new to epigenetics research. I used qiagen software to design primers (forward, biotinylated reverse and sequencing) for my pyrosequencing for methylation analysis. Bisulfite PCR worked well, now I want to pyrosequence amplicons. Two questions:

1. My sequencing primer does no match my bisulfite converted DNA. Is this normal (I am use to primers and DNA sequencing matching)? I do not have access to software anymore, so I can not go back and do analysis again. I am sure there is a simple explanation about this, but I can't find it (this is probably too obvious and hence not explained anywhere)

2. My sequencing primer is too close to my 2 CpG sites of interest, but I can not move primer as amplicon is only 80bp long (lenght of amplicon I can't change). For normal Sanger sequencing, the first 50 bp is rubbish sequence, is it the same for pyrosequencing??

Please help, I am prety desperate




Hi Ursa,

1. The sequencing primer should match your bisulfite modified sequence, unless you are sequencing the other strand? In this case you will be sequencing G/A as opposed to C/T. However as you said you designed the primers with the biotin tag on the reverse strand then I don't think this should be the case. Do you have an CpG sites within your sequencing primer as this may explain why the sequence this different (I think sometimes the software designed primers have an A nucelotide in the sequence covering the C of CpG sites).

2. No it is not the same as sanger sequencing!! It will work from the very first base pair after your sequencing primer. In fact I personally like to start sequencing CpG sites as soon as possible after the primer as I find that reliability can decrease towards the end of longer runs.



Ursa, in addition to HannahxGx's comments, you may not find the sequencing primer matching your bisulfite converted DNA because it is a strand specific PCR amplification, the Qiagen Pyromark software does a really good job with designing primers did you remember what score the software gave the design?

As Hannah alluded to, the primer is most likely to be sequencing the other strand,