Primers for nested-PCR - (Jun/02/2006 )
I am new to methylation work, and I have not done any primer designation before. I have doubts regarding the primers for nested-PCR. All C are supposed to be converted to T during bisulfite treatment ( except methylation CpG), but why the primers designed contain 'G'?
Another question is regarding the treatment with bissulfite. Why does it necessary to digest the DNA before it?
Thanks if anyone can solve this for me.
you are correct about primer design,
primers contain a G because the primer pairs you use are strand specific, ie: they target only one strand of your target loci.
Digestion of genomic DNA reduces complexity and helps with the denaturation process as bisulfite only works on single stranded molecules.
good luck with your foray into methylation work!