Controls for Bisulfite Sequencing - (Feb/05/2007 )
Nick and Krumel
My question is how they were even able to amplify the fragments with those primers??? The primers are based on regions that contain way too many C's that were assumed to be converting to U's ... but in reality all these C's are methylated and won't even convert to U's to start off with... So how can these primers even bind??? wouldn't there be way too many mismatches for the primers to bind???
Krumel, I never thought of your method about using PCR amplified regions as my control...that is an excellent idea...and it will answer some of my question...Thanks.
Nick, Thanks for the info on WGA.
Cheers, Kamelia
Hi Kamelia,
they seem to have been able to amplify solely those templates that had unmethylated C's in the primer binding regions. If the C's in the primer binding sites were methylated, the primers wouldn't bind, that's true. So, the PCR only amplifies the template with unmethylated primers - which makes the whole story suspicious! But how do you know, that the C's in the primer binding site were methylated? To assess their methylation status, you would need other primers amplifying also the binding sites oof the given primers 
Well, like Nick said - rather fishy...
they seem to have been able to amplify solely those templates that had unmethylated C's in the primer binding regions. If the C's in the primer binding sites were methylated, the primers wouldn't bind, that's true. So, the PCR only amplifies the template with unmethylated primers - which makes the whole story suspicious! But how do you know, that the C's in the primer binding site were methylated? To assess their methylation status, you would need other primers amplifying also the binding sites oof the given primers
Well, like Nick said - rather fishy...
Hi Krumel,
I know that the C's in the primer binding site were methylated based on the following paper:
Rossi V, Motto M, Pellegrini L. Analysis of the methylation pattern of the maize opaque-2 (O2) promoter and in vitro binding studies indicate that the O2 B-Zip protein and other endosperm factors can bind to methylated target sequences. J Biol Chem. 1997 May 23;272(21):13758-65.
Which clearly shows the sequence and identifies that these C's are in fact methylated.
I have another question not quite related to this specifically,
In some protocols they phenol/chloroform extract the DNA...In the protocol from another post using agarose beads however they don't, do I have to phenol/chloroform extract my DNA after the use of Restriction enzyme, prior to bead formation and bisulfite treatment?
Thanks, Kamelia
Hi Kamelia,
if you use restriction enzymes you will have to get rid of them prior to bisulfite modification - therefor PC is necessary. Or you shear the DNA by passing it several times through a 21G needle. I have switched to regular use of Eptect Spin Columns and gave up shearing or digestion prior to bisulfite modification, as it didn't affect the outcome using the kit.
K.