MethyLight is a high-throughput, fluorescence-based real-time PCR assay designed to measure DNA methylation. This method allows for the rapid and accurate detection of methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is highly sensitive, quantitative, and reproducible, making it suitable for analyzing DNA methylation patterns in clinical specimens. MethyLight utilizes bisulfite conversion of genomic DNA to create methylation-dependent sequence differences, which are then detected by PCR with primers and a fluorescent probe. The technique can distinguish between mono-allelic and bi-allelic methylation of the MLH1 gene in human colorectal tumor specimens. MethyLight is compatible with very small amounts of DNA and allows for the rapid analysis of multiple gene loci. The study demonstrates that MethyLight can accurately determine the relative prevalence of different methylation patterns and is capable of detecting methylation in human tumors. The assay is also shown to be highly reproducible, even in heterogeneous samples. MethyLight is a powerful tool for the rapid screening of DNA methylation in human tumors, providing valuable biological information that can be associated with the transcriptional silencing of genes relevant to cancer. The technique is particularly useful for analyzing DNA methylation in clinical specimens, as it requires minimal DNA and is compatible with small biopsies and paraffin-embedded tissues. The development of MethyLight should significantly enhance our ability to generate epigenetic profiles of tumor samples, complementing ongoing efforts to determine molecular profiles of tumors using high-throughput genomic and RNA-based technologies.MethyLight is a high-throughput, fluorescence-based real-time PCR assay designed to measure DNA methylation. This method allows for the rapid and accurate detection of methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is highly sensitive, quantitative, and reproducible, making it suitable for analyzing DNA methylation patterns in clinical specimens. MethyLight utilizes bisulfite conversion of genomic DNA to create methylation-dependent sequence differences, which are then detected by PCR with primers and a fluorescent probe. The technique can distinguish between mono-allelic and bi-allelic methylation of the MLH1 gene in human colorectal tumor specimens. MethyLight is compatible with very small amounts of DNA and allows for the rapid analysis of multiple gene loci. The study demonstrates that MethyLight can accurately determine the relative prevalence of different methylation patterns and is capable of detecting methylation in human tumors. The assay is also shown to be highly reproducible, even in heterogeneous samples. MethyLight is a powerful tool for the rapid screening of DNA methylation in human tumors, providing valuable biological information that can be associated with the transcriptional silencing of genes relevant to cancer. The technique is particularly useful for analyzing DNA methylation in clinical specimens, as it requires minimal DNA and is compatible with small biopsies and paraffin-embedded tissues. The development of MethyLight should significantly enhance our ability to generate epigenetic profiles of tumor samples, complementing ongoing efforts to determine molecular profiles of tumors using high-throughput genomic and RNA-based technologies.