Restriction Landmark Genome Scanning

Abstract

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.