70 Epigenetics: Linking Genotype and Phenotype in Development and Evolution, ed. Benedikt Hallgrímsson and Brian K. Hall. Copyright © by The Regents of the University of California. All rights of reproduction in any form reserved. 5 Methylation Mapping in Humans Christoph Grunau The importance of DNA methylation mapping in humans was rapidly recognized once it be- came apparent that 5-methylcytosine (5mC) is not only an exotic and negligible modification of the DNA but an important carrier of epigenetic information. In particular the finding that can- cer is characterized by aberrant methylation in- creased the interest of the scientific commu- nity. Many laboratories attempted to characterize these changes in methylation and to use them as biomarkers for the diagnosis of disease. It had CONTENTS DNA Methylation Mapping Techniques Affinity Purification of Methylated or Unmethylated DNA Fragments Methylation Mapping with Restriction Enzymes Bisulfite Conversion–Based Techniques In Silico Approaches DNA Methylation in the Human Genome CpG Islands Tissue-Specific DNA Methylation DNA Methylation in Reproductive Tissue Imprinted Genes Pseudogenes and Duplicated Genes Repetitive Sequences DNA Methylation in Centromeres, in Pericentromeric and Subtelomeric Regions, and on the Inactive X Chromosome Databases References been known since the 1950s (Wyatt, 1951) that human DNA contains 5mC (later determined to be roughly 1%), and the initial experiments of Vanyushin and his colleagues (1973) and other laboratories showed that DNA of different tis- sues and different developmental stages can ac- tually differ considerably (Table 5.1). Enzymatic digest indicated that in mam- mals (e.g., calf thymus) the majority of 5mC is followed in the 5′–3′ direction by a guanine (Sin- sheimer, 1954). It is today assumed that human