Quantifying % conversion with SsI methytransferase ? - how quantify the amount of C's converted in amplicon of interest (Aug/16/2007 )
I have an amplicon of 1.5kb that i want to fully methylate with SssI methyltransferase. I can do this no problem, but i need the conversion to be over 95% of the C's, and i need to have a VERY precise measurement to ensure convserion.
Bis sequencing is incomplete as not all mC's are converted.
What are my options?
If I understood you correctly, you need to be sure that >=95% of Cs in CG's are methylated by M.SssI?
Then you may try to use tritium labelled AdoMet and compare tritium incorporation by M.SssI into non-methylated and completely methylated (M.SssI + non-radioactive AdoMet) DNA.
How many times do you sss1-treat your amplicon. It may be necessary to have 2 or more rounds for full methylation.
But I am still a little confused - do you need to be sure to have a 100% conversion by bisulfite treatment or full methylation of CpG-C's by Sss1?