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Assessing methylation levels


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#1 spjgjsm

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Posted 11 November 2004 - 09:09 AM

Does anyone have a suggestion for a way to assess quantitatively the methylation levels across a certain genomic region (e.g. a given CpG island)? I am not interested in specific CpG sites, but want to know the amount of methylation in a given genomic region. I am guessing that amplifying the region using PCR and then digesting with methylation-specific enzymes may work....but can this give a quantitative reading?

Cheers

Jon

#2 pcrman

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Posted 12 November 2004 - 01:58 AM

I don't know any current methods that can work that way, but I am certain you can not PCR your DNA and then detect methylation because after PCR, all 5m cytosines will become cytosine. I am currently developing a method which can meaure regional methylation levels. It works in a way similar to ChIP. You first sonicate your DNA into small pieces, and then precipitate it with a 5-methyl-cytosine antibody. After washing, amplify precipitated DNA by genomic PCR. The intensity of the PCR band will tell you the methylation level.

Edited by pcrman, 12 November 2004 - 02:00 AM.


#3 spjgjsm

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Posted 12 November 2004 - 03:55 AM

Hi

Sorry, I meant to say bisulfite treat before the PCR and then digest. But your proposed method sounds interesting - do you have any references or a published protocol we could look at?

Cheers

Jon

#4 pcrman

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Posted 17 November 2004 - 11:32 AM

The method I mentioned is under development and has not been published yet.




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