DNA Methylation in vitro - How to carry out DNA methylation on a given oligo (May/16/2005 )
I am an undergraduate student, working on Alzheimer's disease...we are looking at some genetic regulation that may play a part in this neurodegenerative disease. I will be looking at the APP promoter and looking at methylation/oxidation in control and experimental animals.
My question is as follows:
How can I carry out methylation on an oligo that I have?
I have ordered some oligos for the binding sites of certain transcription factors, and wish to see the effect of methylation on this binding. I, therefore, want to carry out the methylation in the lab.
Please let me know what I can use, and what the protocol might be. I have DNMT1 from New England Biolabs, but have no protocol on how to get this reaction going in vitro.
Please let me know...THANKS!
Why don't just order methylated oligos from vendors such as Invitrogen. I guess you want to induce methylation on the targeted gene as reported in the literature. One colleague of mine tried this without success. I am also skeptical about it because none of those papers appeared in decent journals.
We want to study the effect of oxidative damage (basically 8-oxoG) on the activity of the methylase. So, we will have the G in a CpG island converted to an 8-oxoG by the oligo company, and then want to run the reaction in the lab to see if the methylase works, how its activity is effected, etc. Any ideas on how to do this?
We also want to do this with OGG1, the repair enzyme. Any ideas on that? do you think its possible to get a methylated oligo (i know we can get this) and then run the reaction with Ogg1 in vitro to see its activity?
Hi, there are several techniques you could use, either cutting with a methylation sensitive restriction enzyme or using Tritium-SAM as cofactor. If the oligo is biotinylated, you can bind it to avidin coated plates to purify it (look in pubmed for protocol of Jeltsch + Roth) or you can spot it on whatman paper, wash it and count then (but background is much higher with that method). If you have a scintillation counter the Jeltsch+Roth protocol would be easiest. You could take your biotinylated oligo and add non-oxoG and oxoG as non-biotinylated competitors, and look at the methylation of the biotinylated oligo!
I hope that was the question!