After bisulfite conversion of plant DNA to detect Cytosine methylation, I have certain clones where G bases are changed to A. The process is gDNA extraction, bisulfite conversion (Qiagen Epitect Fast), PCR amplification using degenerate primers, cloning into pGEMT-easy and sanger sequencing.
Hi Joshua,
some question: Is this true for both DNA strands? Do the degenerated primers specifically amplify one bisulfite treated DNA strand or both? Is there a clean signal of the sanger sequencing data at these positions?
can it be that these clones are integrated in the opposite reaction in the vector?
after bisulfite conversion C's are converted to U/T.
on the reverse complement of the PCR product, this means that a CG is seen as a CA (which is the reverse complement of TG)
As Arnoud says, these clones are the reverse strand. During bisulphite-treatment the DNA becomes non-complementary single strands with C converted to T. If this is amplified using CSP, MSP or so on, a new reverse strand is synthesized which has A opposite these converted to T sites. So, reverse strand reads have a seemingly G to A conversion.
Thank you for your replies, yes it appears that the specific primers for the top strand have amplified the bottom strand in a few instances. The signal is clear at all positions in the sequence.
DNA Methylation Data Analysis Workshop
How to use bisulfite-treated sequencing to study DNA methylation
When?
22. - 25. November 2016
Where?
Leipzig, Germany
Link?
http://www.ecseq.com/workshops/workshop_2016-07-NGS-DNA-Methylation-Data-Analysis
http://www.ecseq.com/workshops/workshop_2016-07-NGS-DNA-Methylation-Data-Analysis
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