Do any of the cells have genomic DNA that is 0% methylated? I found scientific reports indicating that peripheral blood mononuclear cells (PBMCs) may exhibit such properties. In my research, I use bovine material and I would like to make a curve for Methylation-Sensitive High Resolution Melting (MS-HRM) analyses by mixing 0% and 100% methylated DNA. Unfortunately, such DNA for cattle is not commercially available. I think, it will be more difficult to obtain 100% methylated genomic DNA.
Hi Robert,
I think no cell type contains fully (genome wide) unmethylated DNA. Several cell types may contain reduced levels e.g. sperm cells. If you are interested in methylation of specific markers you may test several normal cell types for methylation to identify useful unmethylated control. If you want to have a generally unmethylated control you can use genome wide amplification kits. However, we observed low levels of bisulfit-treated WGA-DNA after BS-treatment. Potentially caused by the small size of WGA products.
Regards,
Norman
Hi, how about the mouse ES cell line, which was triple knocked out DNMTs.
The CpG methylation level of the cell line was dramatically reduced.
Article Maintenance of self-renewal ability of mouse embryonic stem ...
DNA Methylation can not be 0% percent in any cell. Reduced methylation in DNMTs KO as Narumi showed is obvious since you are disrupting protein responsible for methylation
Since it is not completely unmethylated, furthermore, what about using such a bisulfate kit, is it inappropriate since the DNA sequence would be replaced?
EpiTect Bisulfite Kit (48)
Cat. No. / ID: 59104
48 EpiTect Bisulfite Spin Columns, Reaction Mix, DNA Protect Buffer, Carrier RNA, Buffers
Thank you for any information. I used WGA to amplify genomic DNA. However, the control methyl-specific PCR within the DNA sequence of interest showed residual methylation which I could not get rid of. So I thought the starting point should be to get DNA with zero methylation. Some of the publications indicated that Peripheral Blood Mononuclear Cells had the lowest possible DNA methylation but removing the whole methylation is difficult. I have to use a shorter section of DNA and do regular PCR. Methylation does not amplify and then I get unmethylated DNA. As for the methylated, I think I will use a methyltransferase that will methylate all cytosines within the selected part of DNA.
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