The objective of this project is the development of a rapid and sensitive assay system for the detection of CpG methylation in tumors and precancerous cells. Methylation of clustered CpG sequences, called CpG islands, is an early and widespread marker of tumorigenesis. The expression of over 60 genes involved in all aspects of cancer is affected by the methylation of CpG islands that overlap with their upstream expression signals. Successful completion of the proposed research will produce a commercial kit that will allow detection of methylation affecting up to 120 CpG islands per patient sample. The resulting diagnostic information will allow for early detection of tumors and precancerous lesions. The RiboMaker(TM) methylation detection kit will be based on signal generation by abortive transcription initiated from CpG sequences associated with selected tumor suppressor genes. RNA polymerase will be programmed to initiate transcription from methylated CpG sequences on a captured target template strand through the use of labeled dinucleotide initiators. Incorporation of labeled chain terminators will allow signal detection by fluorescence resonance energy transfer. A series of potential fluorescence energy donor/acceptor pairs will be tested for incorporation into abortive transcripts using authentic promoters. After appropriate pairs of fluorescent reporters have been defined, they will be used to validate an abortive transcription assay that is based on a model single-stranded template DNA with a known level of CpG methylation.