Patent ID: 8062849

Claim:
A method for determining an increased likelihood of development of breast cancer associated with aberrant methylation of DNA in a subject, comprising: a) co-amplifying DNA in a single subject sample comprising a plurality of different DNA sequences isolated from the subject sample using a mixture of DNA sequence-specific, methylation status-independent outer primer pairs that selectively hybridize to three or more of the DNA sequences under conditions that allow generation of a first amplification product containing first amplicons, wherein the subject sample is selected from the group consisting of ductal lavage fluid, nipple aspiration fluid, breast tissue, breast tumor biopsy, and fine needle aspirates, and wherein further the DNA sequences comprise three or more genes selected from the group consisting of RASSF1A, TWIST, HIN1, and Cyclin D2; b) co-amplifying the first amplicons by real-time PCR under conditions that allow generation of three or more second amplification products; adding three or more members of a set of DNA sequence-specific probes comprising an optically detectable label and three 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; and d) deriving a combined methylation value for the methylation in the subject sample 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 breast cancer in the subject.