Method for quantitative determination of the degree of methylation of cytosines in CpG positions

A method for determining the degree of methylation of a cytosine to be investigated in the genome of at least one cell or one organism, characterized in that the following steps are conducted:

• a) treating a DNA sample with bisulfite and subsequently amplifying a selected fragment of this treated DNA to produce an amplificate;
• b) conducting a sequence analysis of at least two different bases of the amplificate, in which the result is represented in the form of a DNA sequencing electropherogram, wherein the sequence of bases is represented as a curve plot, said conducting step comprising calculating the signal intensity of each base by the area under the peak, standardizing the average signal intensity of at least one, but not all, base types (C, T, A, G) against the average signal intensity obtained for one or more of the remaining base types, and determining the average of at least two positions of this base type within an arbitrarily defined region of the amplificate;
• c) determining a conversion rate at which unmethylated cytosine bases are converted to uracil bases as a consequence of bisulfite treatment, wherein less than all of said unmethylated cytosine bases are converted to uracil following bisulfite treatment, wherein said determining comprises quantitatively comparing the signal intensity of bases which, prior to bisulfite treatment, were unmethylated cytosine bases and which, following bisulfite treatment, exhibited a change in hybridization behavior from that of a cytosine base relative to the signal intensity of bases which, prior to bisulfite treatment, were unmethylated cytosine bases and which, following bisulfite treatment, did not exhibit a change in hybridization behavior from that of a cytosine base, wherein the sense sequence and/or its complementary sequence is used for said determination of the conversion rate;
• d) calculating the actual degree of methylation of the cytosine to be investigated taking into consideration the conversion rate determined in step c).