Patent ID: 8822155

Claim:
A method of diagnosing development of a condition associated with aberrant methylation of DNA in tissue or other DNA sample of a subject, said method comprising: a) co-amplifying DNA in a subject sample comprising a plurality of different DNA sequences isolated from the tissue using a mixture of DNA sequence-specific, methylation status-independent outer primer pairs that selectively hybridize to one or more of the DNA sequences under conditions that allow generation of a first amplification product containing first amplicons; b) co-amplifying the first amplicons by real-time PCR under conditions that allow generation of one or more second amplification products; adding one or more members of a set of DNA sequence-specific probes comprising an optically detectable label and one or more members of a set of DNA sequence-specific methylation status-dependent inner primer pairs, wherein the probes and the inner primer pairs selectively hybridize cognate first amplicons, and wherein the sets of probes and inner primer pairs collectively bind to a plurality of different first amplicons in the first amplification product; c) detecting signal intensity of the optical label 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 tissue of the subject from amounts of methylation determined for the second amplification products as compared with a combined methylation value in comparable normal tissue to diagnose the state of development of the condition in the subject.