Quantitative multiplex methylation-specific PCR
Methods are provided for diagnosing in a subject a condition, such as a carcinoma, sarcoma or leukemia, associated with hypermethylation of genes by isolating the genes from tissue containing as few as 50 to 1000 tumor cells. Using quantitative multiplex methylation specific PCR (QM-MSP), multiple genes can be quantitatively evaluated from samples usually yielding sufficient DNA for analyses of only 1 or 2 genes. DNA sequences isolated from the sample are simultaneously co-amplified in an initial multiplex round of PCR, and the methylation status of individual hypermethylation-prone gene promoter sequences is then determined separately or in multiplex using a real time PCR round that is methylation status-specific. Within genes of the panel, the level of promoter hypermethylation as well as the incidence of promoter hypermethylation can be determined and the level of genes in the panel can be scored cumulatively. The QM-MSP method is adaptable for high throughput automated technology.
1. A kit for determining the methylation status of a plurality of DNA sequences in a DNA sample comprising:
a) a set of DNA sequence-specific, methylation status-independent outer primer pairs that selectively hybridize to one or more of the plurality of DNA sequences under conditions that allow generation of a first amplification product containing first amplicons;
b) a set of DNA sequence-specific, methylation status-dependent inner primer pairs; and
c) 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 hybridize 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,
wherein the DNA sequences comprise three or more genes selected from the group consisting of RASSF1A, TWIST, HIN1, and Cyclin D2.
2. The kit of claim 1 , wherein the DNA sequences further comprise one or more genes selected from RARβ, ESR1, APC1, BRCA1, BRCA2, P16, and HIC1 promoters.
3. The kit of claim 1 , wherein the set of DNA sequence-specific, methylation status-dependent inner primer pairs comprises a subset of the primer pairs that specifically hybridize to methylated first amplicons and a subset of the primer pairs that specifically hybridize to unmethylated first amplicons.