This fast-track SBIR project is to develop a novel multiplexed methylation profiling assay (MMPA) technology for methylation analysis of DNA derived from clinical specimens. Specifically, we will develop the MMPA assays (one for each type of cancer) for analysis of DNA methylation in colorectal, lung, and breast cancer, the three deadliest cancers, respectively. Each assay can simultaneously determine the methylation status of 20 genes in a 10,000 fold excess of unmethylated DNA. The assays can also determine the degree of methylation and the relative abundance of methylated genes in clinic specimens. In addition, the assays will be cost-effective and easy to operate. The goal of Phase I is to determine if the MMPA assay can accurately reveal methylation in DNA derived from clinic samples. Experimentally, we will use our existing MMPA assay to profile the methylation status of eight genes in DNA derived from stools, the most complex specimen. The specific milestone is to demonstrate that this assay can achieve the detection sensitivity and specificity of 90% or better, respectively. Phase II is to develop the MMPA assays for analysis of DNA methylation in colorectal, lung, and breast cancers, respectively. Each MMPA assay """"""""fires one shot"""""""" to """"""""kill four birds"""""""". First, the assay can determine the methylation status of an individual gene;second, the assay profiles DNA methylation among a number of genes;third, the assay has the capability of determining the degree of methylation of methylated genes;and fourth, the assay provides insights into the abundance of methylated genes in clinical specimens. Clearly, these unique features will make MMPA the method of choice for methylation analysis, and thus the success of this project will have a profound impact on both cancer screening and basic cancer research.