Restriction landmark genomic scanning (RLGS) is a method that is employed to analyze the genomes of vertebrates and plants, using restriction enzyme recognition sites as landmarks. The method consists of seven steps: (1) Blocking of the cleaved genomic DNA, to avoid high backgrounds, with nucleotide analogues; (2) Landmark cleavage, with a rare cutting restrictase; (3) Labeling of the cleavage ends, followed by digestion with a higher frequency restrictase; (4) Agarose gel electrophoresis; (5) Digestion with a third restrictase (average fragment size <10 Kb); Polyacrilamide gel electrophoresis; and (7) Autoradiography.
This bidimensional electrophoretic procedure is reproducible and, in this way, whole genome profiles can be generated with mean intervals of ~150 Kb and ~1 Mb, depending on the total genome size. Besides, as each spot represents a locus and its copy number, this technique can detect the hetero- or homozygosity allelic status. Furthermore, as no probes are needed, it could be used to any organism. The authors mentioned that it can be applied to genome mapping, evolutionary distances, polymorphism, etc., but it also has been used to identify imprinted loci, using a combination of methylation- sensitive restriction enzymes. One of those protocols was previously discussed here.
Hatada I. et al. 1991. Proc Natl Acad Sci USA 88:9523-27
epigenetics
