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DNA methylation analysis - direct sequencing gives CT peaks


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#1 MethP

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Posted 13 November 2009 - 01:44 PM

Hello-
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.

#2 methylnick

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Posted 24 November 2009 - 01:00 PM

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




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