I am looking at the possibility of analysing DNA extracted from a very small starting quantity of material (ie. 2 - 3 cells!). I have read up protocols on extracting this DNA, then bisulfite converting, and then whole-genome amplification, but none where the WGA is before the bisulfite conversion. Is this due to bias of standard WGA that would impact methylation results? I am in a situation where I may have access to DNA derived from a couple of (very valuable) cells, but it would already have undergone WGA for some standard genetic testing. If anyone could advise if I can then use this WGAd DNA for methylation analysis it would be very much appreciated.
Thank you!
Most likely your results will be skewed towards unmethylated Cs ;)
If this is a serious question (just checking!) you may need to do some tech development here. For instance, use a polymerase in your amplification procedure that is blocked by Cmet (maybe via RNA pol III) and then try to make it somehow quantitative. Just an idea.
Whole genome amplified DNA is definitely NOT suitable for methylation analysis, because the methylation signature will be completely lost. Whole genome amplification will result in many-fold amplification of the template, but the nucleotides incorporated will not be methylated! So bisulfite treatment of WGA DNA will result in ~100% conversion of the unmethylated C's and the DNA will appear to be completely unmethylated. The only way to preserve the natural methylation pattern is to perform the bisulfite conversion before the whole genome amplification.
As far as I know none of the protocol available for whole genome amplification are able to include the mC where corresponds, so you will miss all the information about methylation status.
If you amplify your DNA prior to biSU, then you will have predominantly if not completely non methylated DNA, because all the amplified material will incorporate non methylated cytosine.
I wouldn´t do that. But there are some BisU protocol that start from only a few ng purified DNA please check
https://www.qiagen.com/us/shop/epigenetics/epitect-fast-bisulfite-conversion-kits/#orderinginformation
Thanks all - I did think it was likely that there would be no preservation of methylation. Thank you!
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