Restriction landmark genomic scanning: analysis of CpG islands in genomes by 2D gel electrophoresis.
Restriction landmark genomic scanning (RLGS) is a method that provides a quantitative genetic and epigenetic (cytosine methylation) assessment of thousands of CpG islands in a single gel without prior knowledge of gene sequence. The method is based on two-dimensional separation of radiolabeled genomic DNA into nearly 2,000 discrete fragments that have a high probability of containing gene sequences. Genomic DNA is digested with an infrequently cutting restriction enzyme, such as NotI or AscI, 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 or AscI sites are frequently used as "landmarks" because NotI or AscI cannot cleave methylated sites and since an estimated 89% and 83% of the recognition sites, respectively, are found within CpG islands. Using a methylation-sensitive enzyme, the technique has been termed RLGS-M. The resulting RLGS profile displays both the copy number and methylation status of the CpG islands. Integrated with high-resolution gene copy-number analyses, RLGS enables one to define genetic or epigenetic alteration in cells. These profiles are highly reproducible and are therefore amenable to inter- and intraindividual DNA sample comparisons. RLGS was the first of many technologies to allow large-scale DNA methylation analysis of CpG islands.
Costello
Joseph F
JF
Department of Neurological Surgery, University of California San Francisco Comprehensive Cancer Center, San Francisco, CA, USA.
Hong
Chibo
C
Plass
Christoph
C
Smiraglia
Dominic J
DJ
D000818:Animals; D001932:Brain Neoplasms / Q000737:chemistry / Q000235:genetics; D018899:CpG Islands; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D016678:Genome; D006801:Humans; D016133:Polymerase Chain Reaction
D004273:DNA, Neoplasm; D004247:DNA; C078382:endodeoxyribonuclease AscI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases