In vitro methylation - How to methylate genomic DNA in vitro (Mar/14/2005 )
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.
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.
Is there any chemical modification possible?
Hi strepto,
labtechie's refferring to this link http://www.protocol-online.org/forums/inde...?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
After SssI treatment, is it necessary to remove the enzymatic reagents (buffer/SAM) by re-precipitating the DNA? or is it sufficient to just heat inactivate it prior to bisulfite treatment?
I phenol-choroform the prep.
make the reaction solution up to 200ul, add 200ul phenol chlroform. Shake, centrifuge and then remove the aqueous phase, precipitate and resuspend for bisulfite treatment.