Methods and systems for detecting methylation changes in dna samples

A method for sensitive detection of methylation changes in a DNA sample, the method comprising:

• (a) providing a DNA sample, wherein the DNA sample contains less than 5% single-stranded DNA (ssDNA);
• and
• (b) subjecting the DNA sample to methylation ratio analysis by digesting the DNA sample with at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease, co-amplifying from the digested DNA at least one restriction locus differentially methylated between normal and disease DNA and at least one control locus in a single reaction mixture, and comparing a ratio between signal intensities of the amplification products of each of said at least one restriction locus and the control locus to at least one reference ratio,
• thereby detecting methylation changes in the DNA sample at a detection sensitivity of at least 1:100.

A system for detecting methylation changes in a DNA sample, the system comprising:

• (a) a DNA sample, wherein the DNA sample contains less than 5% single-stranded DNA (ssDNA);
• and
• (b) components for carrying out a methylation ratio analysis, comprising:
• (i) at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease for digesting the DNA sample;
• (ii) a plurality of primer pairs for co-amplification of a plurality of genomic loci in a single reaction mixture from the DNA sample following digestion, wherein the plurality of genomic loci comprises at least one restriction locus differentially methylated between normal and disease DNA and at least one control locus;
• and (iii) computer software stored on non-transitory computer readable medium, the computer software directs a computer processor to determine methylation changes in the DNA sample based on a comparison of a ratio of signal intensities of the restriction locus and the control locus following amplification to a reference ratio,
• with the methylation changes determined at a detection sensitivity of at least 1:100.