detection of 5-methyl cytosine (bisulfite method)

[Onno Myriam]

I ll tried to use an improved method for detection of 5-methyl cytosine by PCR-based genomic sequencing (Biotechniques Vol16 410-417 1994).
This method is based on bisulfite modification of genomic DNA under conditions whereby cytosine is converted to uracil but 5-methyl cytosine remains unaltered.
I have three questions: Is it necessary (possible) to autoclave the bisulfite/hydroquinone solution ? what is exactly the method to alkali denature genomic DNA ? After bisulfite treatment, is it necessery to add NaI before the incubation with the Glassmil
k?

	Any answer is welcome

	Thanks

	
	Myriam

Onno Myriam
Laboratory of Haematology
University of Rennes I
France
e-mail: Onno.Myriam at univ-rennes1.fr