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HPLC protocol for methylation quantification - (Nov/12/2005 )

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Hi all,

I'm about to start investigating DNA methylation with an LC method and have some questions:
* Is denaturation of the DNA really necessary? Can this influence the methylation?
* Is digestion with DNAse necessary before digestion with nuclease P1?

Greetings & thanks in advance for any help,



Hi Michiel,

Denaturation of DNA prior to using nuclease P1 is essential as the enzyme can only act on single stranded substrates. I do a 5 minute 99.9 degree hold then put my samples on ice, allow to cool, then add the nuclease P1. I'm very doubtful as to whether this affects methylation.

I don't use DNAse I, however if you were using a larger sample of DNA then it probably wouldn't hurt. At the moment I use 3ug of DNA with about 1.5 units of Nuclease P1 from US Biologicals without DNAse treatment.

Best of luck with your work!


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