Assay for the detection and quantitation of hemimethylation

A method to determine the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide sequence within a palindromic CpG methylation site, in a pool of DNA molecules having mixed DNA methylation states at that palindromic CpG site, comprising:

• obtaining an isolated genomic DNA sample;
• digesting the DNA sample with an excess of a methylation-sensitive restriction endonuclease that cleaves according to a recognition sequence motif comprising a palindromic CpG methylation site, whereby the endonuclease cleaves the DNA at a specific recognition motif position if the methylation state of the respective palindromic CpG methylation site is un-methylated, but not hemi-methylated or fully-methylated;
• treating the endonuclease-digested DNA with bisulfite, whereby unmethylated cytosine residues on both strands of the DNA are converted to uracil;
• amplifying one strand of the bisulfite-treated digested DNA with primers flanking the specific recognition motif position comprising a bisulfite-treated specific CpG dinucleotide of the palindromic CpG methylation site, whereby any uracil residues are converted to thymidine residues;
• and
• determining, by sequence analysis of the amplification products or of individual clones thereof, the presence of either cytosine or thymidine at the position corresponding to the first nucleotide position of the specific CpG dinucleotide, whereby the fraction of DNA molecules that is hemi-methylated at the specific CpG dinucleotide is determined by averaging results obtained by the sequence analysis.