Quantitative multiplex methylation-specific PCR

A method of diagnosing development of a cancer associated with hypermethylation of CpG islands in cancer-associated tissue of a subject, said method comprising:

• a) co-amplifying CpG islands in a subject sample comprising a plurality of different DNA sequences isolated from the carcinoma-associated tissue using a mixture of DNA sequence-specific, methylation status-independent outer primer pairs that selectively hybridize to cognate DNA sequences under conditions that allow generation of a first amplification product containing first amplicons;
• b) using real time PCR to co-amplify CpG islands in the first amplicons under conditions that allow generation of one or more second amplification products, using one or more members of a set of DNA sequence-specific probes comprising one or more fluorescent moieties and one or more members of a set of DNA sequence-specific methylation status-dependent inner primer pairs that selectively hybridize to one or more first amplicons, wherein the sets of probes and inner primer pairs collectively hybridize to a plurality of different first amplicons in the first amplification product;
• c) detecting fluorescence due to the presence of the fluorescent moieties in the second amplification products to determine the amount of methylation of the CpG islands therein, wherein sensitivity of detecting methylated versus unmethylated amplicons is at least 1 in 100,000;
• and
• d) deriving a combined methylation value for the CpG islands in the DNA sequences from amounts of methylation determined for the second amplification products as compared with the combined methylation value in a comparable normal DNA sample to diagnose the state of development of the cancer in the subject.

A method for determining the methylation status of a DNA sample comprising:

• a) co-amplifying a plurality of DNA sequences obtained from a subject sample using a mixture of DNA sequence-specific, methylation status-independent outer primer pairs that selectively hybridize to one of the DNA sequences under conditions that allow generation of a first amplification product containing first amplicons;
• b) using quantitative PCR to amplify first amplicons under conditions that allow generation of second amplification products, using a set of DNA sequence-specific, methylation status-dependent inner primer pairs and a set of DNA sequence-specific probes comprising one or more distinguishable optically detectable labels, wherein a combination of inner primer pair and probe selectively hybridizes to one first amplicon, and wherein the sets of inner primer pairs and probes collectively hybridize to a plurality of first amplicons in the first amplification product;
• c) detecting signal intensities of the one or more distinguishable labels in the second amplification products to determine the amount of methylation of the second amplification products, wherein sensitivity of detecting methylated versus unmethylated amplicons is at least 1 in 100,000;
• and
• d) deriving a combined methylation value for the methylation in the DNA sequences from amounts of methylation determined for the second amplification products as compared with the combined methylation value in a comparable normal DNA sample to determine the methylation status of the DNA sample.