Restriction Landmark Genome Scanning
- 989 Downloads
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.
KeywordsWhatman Paper Restriction Landmark Genomic Scanning Buffer Chamber Restriction Trapper Stopcock Valve
- 6.Kuromitsu, J., Yamashita, H., Kataoka, H., Takahara, T., Muramatsu, M. Sekine, T. et al. (1997) A unique downregulation of h2-calponin gene expression in Down’s syndrome: a possible attenuaton mechanism for survival by methylation at the CpG island in the trisomic chromosome 21. Mol. Cell. Biol. 17, 707–712.PubMedGoogle Scholar
- 8.Okazaki, Y., Hirose, K., Hirotsune, S., Okuizumi, H., Sasaki, N., Ohsumi, T., et al. (1995) Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the spot-bombing method, Proc. Natl. Acad. Sci. USA 92, 5610–5614.PubMedCrossRefGoogle Scholar
- 16.Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1990) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar