Restriction Landmark Genome Scanning

Restriction landmark genomic scanning (RLGS) is a method that provides both a quantitative genetic and epigenetic (cytosine methylation) assessment of thousands of CpG islands in a single gel without prior knowledge of gene sequence (1 ). The method is a two-dimensional separation of radiolabeled genomic DNA into nearly 2,000 discrete fragments that have a high probability of containing gene sequences and are ideal in length for cloning and sequence analysis. Genomic DNA is digested with an infrequently cutting restriction enzyme such as NotI , radiolabeled at the cleaved ends, digested with a second restriction enzyme, and then electrophoresed through a narrow, 60 cm-long agarose tube-shaped gel. The DNA in the tube gel is then digested by a third, more frequently cutting restriction enzyme and electrophoresed, in a direction perpendicular to the first separation, through a 5% nondenaturing polyacrylamide gel, and the gel is autoradiographed. Radiolabeled NotI sites are frequently used as “landmarks” because Note can not cleave methylated sites and since an estimated 89% of NotI sites are within CpG islands (2 ). Using a methylation-sensitive enzyme, the technique has been termed RLGS-M (3 ). The resulting RLGS profile displays both the copy number and methylation status of the CpG islands. These profiles are highly reproducible and are therefore amenable to inter- and intra-individual DNA sample comparisons.