I am trying to optimize a protocol to investigate the differential methylation in gene promoter.
I am using published primers for the OPRM1 promoter for nested PCR on bisulfite converted DNA (extracted from blood). I am able to obtain single band from my PCR and proceed with BigDye Sequecing of the PCR product.
However, I am having problems getting consistent seqeuncing results.
1)I find that the % methylation from the same PCR product when sequenced with forward and reverse primers to vary significantly~ up to 20-30% difference. I believe that my DNA is fully converted as I did not observe any non CpG C's in the sequences (no partial C>T conversion observed in these areas).
2) When I repeat the BigDye sequencing on the same PCR product, I am not able to reproduce the % methylation observed. However, I do notice that the reverse primer tend to give a more consistent result. The sequencing with forward primer shows really big variation. I do note that some papers prefer to use reverse primers for bisulfite sequencing. does anyone know why is it so? is there a reason why the reverse primer is more reliable?
Also, as the first~80bp of sequeing results are usually not very reliable, how can one rely on only sequencing with either one of the primers ? espcecially when longer PCR product size is not recommended for bisulfite sequencing.
3) I believe that my DNA is fully converted as I did not observe any non CpG's C in all my sequencing results (no C minor peaks observed). However, when I repeated the bisulfite conversion, followed by nested PCR and BigDye sequencing on the same DNA sample, I observed huge variation in the % methylation observed as well.
Does anyone have similar problems and could suggest to me what could I have possibly done wrong? Please help!!
Thank you in advance.