Global DNA methylation - [3H]dCTP method - (Apr/16/2007 )

Hello everybody,

Did anybody try to determinate global DNA methylation by method described in Biochemical and Biophysical Research Communications 262, 624–628 (1999) - Pogribny et al.?
This method is based in [3H]dCTP incorporation after DNA cleavage with Hpa II restriction enzyme.
How did you wash the filter after transfer the samples?
Is this the better method for global methylation?

Thank you very much
Regards
Anna

Hi Anna,

this method works fine, however, you depend on the quality of HpaII/MspI digestion. A non-radioactive adoption of the method can be found here (PMID: 15565495).

Krümel

Thanks Krümel.
Actually my boss really wants to determinate by this method because we already bought the [3H]dCTP.
I'm having some trouble with standardization. Could anyone help me, please?
How can you control the HpaII / Msp digestion?
How do you wash the filter after transfer the samples to ion-exchange membrane?
Is there a specific time to read the samples after add the scintilation liquid?

Thanks