Methods for detecting genomic DNA methylation

A method for determining the methylation status of one or more genomic DNA loci, wherein the method comprises:

• (a) isolating genomic DNA from one or more cells of a biological sample of a subject;
• (b) digesting a first sample of the genomic DNA with a methylation-insensitive restriction enzyme to form a first sample of genomic DNA fragments, and digesting a second sample of the genomic DNA with a methylation-sensitive restriction enzyme to form a second sample of genomic DNA fragments;
• (c) ligating a nucleic acid linker to the genomic DNA fragments of the first sample and the second sample;
• (d) amplifying the first sample and the second sample of genomic DNA fragments to generate a first sample and a second sample of amplified DNA fragments respectively;
• (e) contacting the amplified DNA fragments of the first sample and the second sample with microspheres, each microsphere having a unique fluorescent label and a genomic DNA locus-specific probe, wherein the amplified DNA fragments hybridize to the probe;
• (f) quantifying the amplified DNA fragments of the first sample and the second samples bound to microspheres by flow cytometry;
• and
• (g) comparing the amount of hybridized DNA fragments in the second sample to the amount of hybridized DNA fragments in the first sample to determine the methylation status of the genomic DNA locus that corresponds to the locus-specific probe;
• wherein the digesting and ligating are performed in a single reaction, and
• wherein, upon ligation with the nucleic acid linker, the genomic DNA fragments do not have a restriction site for the methylation-sensitive enzyme and the methylation-insensitive enzyme.