from: Epigenetics: A Reference Manual (Edited by: Jeffrey M. Craig and Nicholas C. Wong). Caister Academic Press, U.K. (2011)
A large variety of methods to measure DNA methylation have been developed and used extensively over the last twenty years. These have been based on selective restriction digestion of methylated DNA, the capture of methylated DNA by methyl binding proteins or antibodies, or bisulphite conversion of DNA. However, all restriction enzyme based methods are dependent on available restriction sites for methylation-specific restriction enzymes and therefore cannot be used to analyse every CpG site in the genome. Although immunoprecipitation methods are not sequence specific, they are unable to provide methylation data at a single-base resolution. Therefore, both of these approaches are limited in their applicability. On the other hand, bisulphite conversion based methods allow methylation studies directed to any CpG site in the genome. Sodium bisulphite treatment of DNA converts all unmethylated cytosines into uracil while methylated cytosines remain unchanged, thus transferring an epigenetic difference into a measureable genetic difference. A variety of downstream methods such as Polymerase Chain Reaction (PCR), sequencing, Single Nucleotide Polymorphism (SNP) genotyping and mass spectrometry can be coupled with bisulphite conversion. This chapter provides an overview of some of these methods, concentrating on bisulphite sequencing, methylation-specific PCR, pyrosequencing, MassARRAY EpiTYPER and Infinium HumanMethylation27 BeadChip. Considerations for assay selection and detailed protocols for each method are presented in the end of this chapter read more ...