Quantitative multiplex methylation specific PCR method—cMethDNA, reagents, and its use

A method of diagnosing the development of a disease associated with aberrant DNA methylation in a DNA-containing sample from a subject comprising:

• a) adding to the sample containing DNA from the subject, a plurality of copies of a plurality of synthetic polynucleotide standards (STDgenes), wherein each different STDgene is specific for a different TARGETgene of interest, wherein each different STDgene comprises a DNA sequence having a similar number of nucleotides as the TARGETgene for which it is specific, wherein each different STDgene has 5′ and 3′ end nucleotide sequences which are homologous to nucleotide sequences of the TARGETgene for which it is specific, wherein the intervening DNA sequence between the 5′ and 3′ ends of each different STDgene lacks homology to the sequence of the TARGETgene for which it is specific or other human DNA;
• and wherein at least two different STDgenes are added to the sample;
• b) isolating and processing the DNA in the sample of a) to prepare the DNA for methylation-specific PCR via bisulfite treatment;
• c) amplifying the DNA in b) with at least two different external methylation-independent PCR primer pairs, where each different primer pair is capable of selectively hybridizing to only one of the TARGETgenes of interest and also to the STDgene specific for that one TARGETgene, using PCR under conditions sufficient to produce a first amplification product;
• d) obtaining a diluted sample of the first amplification product of c);
• e) for each different TARGETgene of interest adding to d) one pair of internal methylation-dependent PCR primers specific for the TARGETgene of interest, one pair of internal PCR primers specific for the STDgene for the TARGETgene of interest, a unique optically detectable labeled DNA probe which specifically hybridizes to the TARGETgene of interest, and a unique optically detectable labeled DNA probe which specifically hybridizes to the STDgene for the TARGETgene of interest;
• and
• f) amplifying the DNA in e using quantitative real-time PCR under conditions sufficient to produce a second amplification product which provides quantification of the amount of copies of (i) the methylated TARGETgenes of interest and (ii) the STDgene for the TARGETgenes of interest, wherein an increase in the quantity of methylated copies of the TARGETgenes of interest obtained from the sample compared to a control is indicative, of an increased risk or presence of the disease;
• and
• g) diagnosing the subject as having an increased risk or presence of the disease.