I am trying to get sequences from candidate genes in Drosophila. I have sequences after bisulfite treatment and cloning and confirmed that there is methylation present.
I want to compare males and females using direct sequencing to see if there is quantitative differences in the whole body at certain base positions.
My problem is that although the sequencing worked in the females, in males it did not. I suspect that it is because I have methylation in the primer site and I have discovered (from the clones) that CpAs, CpGs, CpTs and CpGs are all methylated in my flies. This makes it hard to design one primer set between males and females that amplifies treated sequences.
Does anyone have a suggestion for going about this another way? For example, off the top of my head, could I perform PCR with an equal mix of differing primers that would amplify differently methylated (in the primer site) sequences that would be comparable between males and females?
Any suggestions welcome, drosophila are a bit of a pain for bisulfite sequencing!
Thanks in advance.
Bisulfite sequencing in Drosophila - need ideas!
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