Like many other scientists on this forum I'm having some issues with bisulfite conversion of genomic DNA.
I am using the EZ DNA methylation kit by ZYMO and I use control 2 control samples, one fully methylated and one fully non-methylated (also by ZYMO). My PCR primers are specific for converted DNA so they bias my PCR towards the converted DNA. Then I use the SNaPshot reaction to interrogate specific CpG sites for their methylation status.
My PCR works, although not optimal, but I get the amplicons that I expect. When I then perform the SNaPshot on my non-methylated control sample it seems like the conversion is incomplete, some cytosines (in CpG order) remained cytosine and some seem partially converted. So then I decided to sequence my amplicons because I did not understand why my PCR worked on the partially converted DNA.
The sequence then shows that (for the non-methylated control) all the C's that are not CpG are fully converted, which explains why the PCR works, but the C's in CpG order are not completely converted.
Could it be that, even though the cytosine is non-methylated, a cytosine in a CpG dinucleotide is still harder to convert by bisulfite?
Or is it possible that the non-methylated control is actually not fully non-methylated?
I am a bit lost at the moment!
Have any of you seen this phenomenon before?
I hope someone can help me