DNA methylation analysis - direct sequencing gives CT peaks - (Nov/13/2009 )
I am completely new in the field of bisulfite sequencing and have some questions concerning my first analysis of the obtained sequence reactions. I have used the DNA methylation KIT from Zymo to convert my gDNA and designed primers for subsequent amplification using MethPrim. Up to that timepoint it has worked fine. Then I started sequencing and received low quality sequences. I have designed internal primer in order to improve the quality. It helped a bit.
Now my questions:
- do I need to do gel extraction on all of them?
- is cloning an option?
- how often do I have to convert the DNA/ amplify in order to confirm my findings.
I have many C-T double peaks which (as far as I understood) could be explained by either a mixture of cells or two alleles differentially methylated. Strange enough: in my case one allele is deleted and I want to check if the other one is silenced by methylation. gDNA has been extracted from cancer cells (assumed to be >90%). So is that an indication that my double peak results from contamination with another sample or is there another explanation?
Thanks a lot for your help.
another explanation is that your bisulphite conversion was not 100% efficient and coupled to poor primer design (that does not target fully converted DNA) you are getting a mixed population of amplicons of various conversions coming through