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how to measure global methylation changes


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#1 pufalicious

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Posted 15 September 2004 - 05:08 PM

Hi,
I'm not at all experienced in the methylation field and am trying to figure out if DNA from a treated cell population is less methylated than a non-treated cell population. Since I'm not interested in exact quantification at this point, I decided to simply digest my DNA with MspI and HpaII and run the unamplified DNA on a 0.8-1% agarose gel. What I would like to know is how reliable is this method? I've tried this method twice on the same samples of DNA and have had different results.

Are there any other easy and quick ways to look at global methylation changes?

Thanks!

#2 pcrman

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Posted 17 September 2004 - 04:45 PM

For quantitation of global methylation, you can use the SssI methyltransferase assay. SssI methyltransferase is able to catalyase the de novo methylation of CpG sites using the universal methyl donor SAM. You label SAM with tritium and then determine how much tritium can be transferred to your DNA. The more tritium transferred, the less content of 5-methyl cytosine in your DNA.

There are other methods for detecting global methylation such as immunoassays, chromatography.




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