4 replies to this topic

[strepto]

Hi,
This is a query:
How to methylate normally unmethylated genomic DNA in vitro?
Is there any chemical modification possible?
If we can do it in vitro it may help us standardizing Methylation specific PCR where we can be sure that the DNA is methylated.
Because sometimes the DNA may not be really methylated if we purify the genomic DNA from any source which we assume to be methylated.  And trouble shooting becomes difficult when we don't get a product.
We are in the begining in this field.
Just curious.
Thanks.

[labtechie]

Hi-- Look under "the actual bisulfite treatment," where methylnick posted some amazing suggestions about how to generate and test a positive control using M Sss I methylase.  I'm still figuring out how to do it myself, but maybe Nick can help us both out.

[methylnick]

strepto, on Mar 15 2005, 12:29 AM, said:

How to methylate normally unmethylated genomic DNA in vitro?
Is there any chemical modification possible?

Hi strepto,

labtechie's refferring to this link http://www.protocol-...?showtopic=4329 here.

There is an enzyme available that than in-vitro methylate DNA it is called SssI methylase and is available from NEB.

I haven't methylated genomic DNA myself(only tried it in plasmids) but I can't see why the enzyme would not methylated gDNA.

Have a look at the link. It's always nice to have a positive methylation control, although to obtain a negative methylation control.......that may be a little challenging becuase the demethylase enzyme has still not been found....

Good luck!

Nick

[pcrboy]

methylnick, on Mar 15 2005, 03:13 PM, said:

It's always nice to have a positive methylation control, although to obtain a negative methylation control.......that may be a little challenging becuase the demethylase enzyme has still not been found....

methylnick, i do believe the demethylase has been identified in arabdopsis and zebrafish. there have been rumors (which have been confirmed) that several groups have isolated the mammalian demethylase. however, whether it is functional or whether it plays a critical role in vivo is unknown. the demethylase can actively demethylate DNA in vitro.

[methylnick]

Hi PCR boy,

indeed you are correct, this work was presented in a recent keystone meeting and it's fascinating, I look forward to the knockout studies of this gene to see if indeed you get failure of methylation erasure.

Nick