Technologies for the identification and quantification of methylated cytosines in DNA that yielded important information about patterns of methylated DNA were developed during the 1950s and 1960s. However, none of these alone could define the distribution of methylated cytosines in eukaryotic DNA or advance our understanding of its function.35 With the advent of molecular biology, assays capable of sequencing genomic DNA and localizing genotypic differences in genomic targets evolved rapidly in response to demands in research and medicine, and restriction endonucleases were an integral component of many of these assays. Typically restriction endonucleases are bacterial enzymes that recognize palindromic sequences four to eight bases long and make sequence-specific cuts in the phosphate-pentose backbone of DNA to yield ''restriction fragments'' of the molecule.
Several restriction enzymes include CpG in their recognition sequence such as Hpall (CCGG), Mspl (CCGG), Aval (CPyCGPuG), Sail (GTCGAC), and Smal (CCCGGG). lnterestingly, some of these enzymes, such as Hpall, do not cut the DNA if the CpG sequence is methylated, while others, such as Mspl, cut the DNA regardless of the methylation state.36 lnvestigators took advantage of this differential property to determine the pattern of methylation in specific regions of DNA.
ln 1975, Edwin Southern combined the specificity of restriction enzymes with slab gel electrophoresis to identify sequence variation in fragments of DNA.37 He used restriction enzymes to generate predictable fragmentation of genomic DNA, and gel electrophoresis to separate and array the DNA fragments by size. Waalwjik and Flavell38 used Southern's technique to cut total rabbit genomic DNA with either Hpall or Mspl followed by agarose electrophoresis, Southern blotting, and hybridization to a 32P-labeled globin probe. Other investigators applied Southern's technique in experiments similar to that of Waalwjik and Flavell virtually simultaneously. This series of elegant experiments revealed a definitive pattern of methylated cytosines in somatic DNA, that both strands of DNA contained methylated cytosines, and that these patterns were maintained through DNA replication (reviewed in Razin and Riggs35). Waalwjik and Flavell were first to demonstrate the pattern and location of methylated cytosines at CpG residues in DNA. Additional studies indicated that unmethylated DNA sequences generally remained unmethylated, and that methylated sequences retained their methyl moieties for at least 50 generations of growth and culture. ln this way, the clonal inheritance of the DNA methylation patterns was established; the tissue specificity of these patterns provided further support for this conclusion.
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