Dual-probe digital droplet PCR strategy for specific detection of tissue-specific circulating DNA molecules

A method of analyzing the methylation status of methylation sites of a double-stranded DNA molecule which comprises at least two methylation sites per single strand of said double-stranded DNA molecule, the double-stranded DNA molecule being comprised in a specimen, the method comprising:

• (a) contacting the double-stranded DNA with bisulfite to generate single-stranded DNA molecules of which demethylated cytosines of said single-stranded DNA molecules are converted to uracils;
• (b) fractionating the specimen into a plurality of specimen fractions wherein more than 50% of the fractions contain no more than one single-stranded DNA molecule per specimen fraction;
• (c) contacting said single-stranded DNA with amplification primers under conditions that generate amplified DNA from said single-stranded DNA;
• and
• (d) determining the methylation status of said at least two methylation sites of said single-stranded DNA molecule in at least one of said specimen fractions, wherein a methylation status of each of said at least two methylation sites on said single-stranded DNA molecule is indicative of the methylation status of methylation sites of a double-stranded DNA molecule;
• wherein said determining comprises contacting said amplified DNA with:
• (i) a first probe that hybridizes to said amplified DNA at a site which comprises the first of said at least two methylation sites;
• and
• (ii) a second probe that hybridizes to said amplified DNA at a site which comprises a second of said at least two methylation sites, wherein said first probe and said second probe are labeled with non-identical detectable moieties.