Identification of imprinted loci by methylation: use of methylation-sensitive representational difference analysis (Me-RDA).

The technique of representational difference analysis (RDA) was originally described by Lisitsyn et al. (1993) as a means of identifying differences between complex genomes by the use of subtractive hybridisation (1). This protocol for methylation-sensitive RDA (Me-RDA) describes adaptations to the original technique that, by the use of oligonucleotides for HpaII or Hin6I sites, allow the identification of sequences whose methylation differs between two sources of DNA. Differences in the methylation of the maternal and paternal alleles of imprinted genes have been described for most imprinted genes studied to date (2). The importance of methylation in the control of imprinted gene expression has been clearly demonstrated by the perturbation of allelic expression in mice deficient in Dnmt1, the major mammalian DNA methyltransferase (3), and methylation has been suggested to fulfill the necessary requirements of an "imprint" (4).