This study reports a novel high-throughput methylation assay that utilizes highly sensitive and accurate fluorescence-based real-time PCR (TaqMan®). We have shown that MethyLight is not only highly specific, sensitive and reproducible, but that it also can rapidly detect biologically relevant information in patient samples. MethyLight is a PCR-based method that requires only minute amounts of DNA of modest quality, making it compatible with small biopsies and paraffin-embedded tissues.
In this initial study, we have not explored all variants of the MethyLight assay as outlined in Figure . In particular, application B promises to be a powerful method, since it has the potential to provide quantitative information on the relative prevalence of different sequence variants, representing different methylation patterns, in a pool of PCR products. However, a truly quantitative application of version B in Figure will require a systematic exploration of hybridization efficiencies of probes with varying numbers of CpG mismatches. We expect some cross-reactivity to occur in cases of single mismatches in relatively long oligonucleotide probes. The relatively simple version of MethyLight technology explored here (applications A and D), though less comprehensive, is more cost-effective and capable of rapidly generating biologically relevant information with a minimal amount of manual labor, as shown in Figure .
It should be emphasized that the MethyLight technique was not designed to yield high-resolution methylation information, such as the pattern information obtainable with bisulfite genomic sequencing (Fig. ) or the accurate methylation percentage determination at single CpGs obtainable with COBRA (Fig. ). Rather, its strongest features are its high-throughput capabilities and its high degree of sensitivity. The assay as we have explored it here is also highly quantitative, but in a different way than traditional methylation analysis methods are. Rather than capturing all methylation occurences of a CpG dinucleotide in a heterogeneous genomic DNA sample, the MethyLight technique can very accurately determine the relative prevalence of a particular pattern of DNA methylation. However, in doing so, the technique is oblivious to all other methylation permutations. MethyLight is similar in this respect to MSP (5
), but differs in that it determines the relative amounts of a particular methylation pattern with quantitative accuracy. MSP is an endpoint analysis technique, whereas the quantitative nature of MethyLight is based on the cycle number at which the fluorescent signal crosses a threshold in the exponential phase of the PCR reaction. This allows quantitative conclusions to be drawn concerning methylation levels relative to a control reaction as shown for MLH1
in Figure . This would not have been possible with a standard MSP reaction.
The most striking advantage of MethyLight, as compared to existing techniques, is its potential to allow the rapid screening of hundreds to thousands of samples. Unlike other techniques, the MethyLight assay is completed at the PCR step, without the need for further gel electrophoretic separation or hybridization. This reduces the chance of sample contamination and error, and dramatically decreases the amount of labor involved in DNA methylation analysis. The technique is also extremely rapid. We recently reported a relatively small-scale MethyLight analysis of four CpG islands in 50 human tissue samples, for a total of 200 methylation analyses (8
). The methylation analysis for this study was accomplished in <3 days, including the bisulfite conversion step. Once the sodium bisulfite conversion step has been performed, a complete determination of the methylation status can be obtained in <2 h of 96 separate gene loci for a single sample, or of a single locus in 96 separate DNA samples. The development of this technique should considerably enhance our ability to generate epigenome maps of tumor samples. As such, it should extend and complement ongoing efforts to determine molecular profiles of tumor samples using high-throughput genomic and RNA-based technologies.