Bisulfite sequencing -- Positive control? - bifsulfite sequencing (Apr/28/2005 )
I have been using Zymo's EZ methylation kit, and have been wondering for some time how efficient the conversion from unmethylated C to T was. Do you guys know of any good positive controls to test the conversion efficiency.
I thought about PCR amplifying a gene (in principle, all cytosine should be unmethylated following PCR), and then bisulfite treat those, clone and sequence. So I should see all cytosines converted to Ts. Am I missing something? Any ideas?
any help would be appreciated.
That sounds like a good plan.
Bisulfite conversion, like many enyzmatic reactions is never 100% efficient. Thus primer design is cruicial to obtain fully converted templates.
After PCR you will lose the methylation marks on the gene and then you can bisulfite treat that. There is literature about the technique of bisulfite conversion and the efficiencies but which ones they are elude my memory at this time.
It sounds like a good idea, but there may be a small problem. PCR products are short fragment of DNA without any methylation. 100% C->U conversion of PCR products doesn't necessarily mean the same conversion efficiency with genomic DNA which is of higher molecular weight and harder to be denatured to single stranded. Plasmid DNA may be a better choice.
That's a very good point pcrman!