Purpose of Genomic DNA hydrolysis - (Apr/13/2008 )

Hi,

I m new to DNA methylation studies. I read some papers' Materials & Methods, they perform genomic DNA hydrolysis using nuclease P1 and alkaline phosphatases. I have searched online but there is not much info. so I'd like to ask you experts what is the purpose of this hydrolysis step and the uses of the 2 respective enzymes?

Thx in advance.

Sounds to me like you are looking at papers on genome-wide methylation levels. Nuclease P1 digests DNA into component nucleosides, ie A/C/T/G/methyl-C. Alkaline phosphatase removes the phosphate group from the nucleosides. These steps are performed before you carry out HPLC, and then after HPLC you can quantitate the percentage of methyl-cytosine present on the DNA.

I think it is explained well in this paper:

Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography
B.H. Ramsahoye
Methods 27 (2002), 156-161