DNA methylation is the most widely studied epigenetic modification to DNA. DNA methylation occurs almost exclusively at the cytosine residue in CpG dinucleotides in many organisms. It is becoming increasingly evident that DNA methylation plays a critical role in development, gene regulation, chromosome stability and the onset and progression of disease. Thus, knowledge of the DNA methylation state of the genome is essential. Although a variety of methods are available to assess DNA methylation status, the study of DNA methylation is compromised by low sensitivity, laborious protocols, excessive time and expensive reagents. While restriction enzyme techniques typically require a large amount of material and do not assay all CpG dinucleotides in the regions of interest, bisulfite mutagenesis/cloning/sequencing technology, which reduces the amount of material and provides a more comprehensive analysis of the regions of interest, is time consuming and low throughput. Pyrosequencing is a fast, simple and quantitative method for analysis of CpG methylation in multiple sites in a single assay. It eliminates the requirement for CpGs in restriction enzyme sites, large amounts of DNA and laborious protocols. It also less prone to bias artifacts generated when traditional bisulfite mutagenesis, cloning and sequencing techniques are used and provides a more quantitative assessment of DNA methylation status. The PyroMark Q96 MD is a highly sensitive and quantitative genetic analysis system based on Pyrosequencing technology. Sample preparation and analysis are very fast and the PyroMark Q96 MD is high throughput: 96 samples can be assayed in less than 1 hour after PCR. Furthermore, this instrument and technology will allow the determination of DNA methylation status in the presence of SNP's, thereby facilitating the assay of allele-specific methylation patterns of imprinted genes as well as the assay of DNA methylation of disease associated genes in combination with polymorphisms. This application describes a wide array of uses for the PyroMark Q96 MD at the University of Pennsylvania and other Philadelphia area institutions. The uses include allele-specific DNA methylation analysis of imprinted gene regulation in animal models, assisted reproduction technology conceptuses and human disease. DNA methylation will also be examined in association with stress and behavior, cancer, environmental exposures, intrauterine growth retardation/low birth, diet-induced obesity, autism and aging. The technology afforded by the PyroMark Q96 MD will allow a more complete, accurate and efficient understanding of the role of DNA methylation changes in development, behavior, environmental exposures and disease.
