Figures & data
1 Retinoblastoma gene (RB1) containing 27 exons and the 6.1-kb SacI fragment with three (distinguishable) SacII restriction enzyme cutting sites, three SmaI cutting sites, and probe p123 that spans these restriction enzyme sites. The leftmost SacII circle encloses two SacII sites, which are separated by 23 bp only. Circles = SacII sites; triangle = SmaI sites.
2 Southern blot analysis of tumor DNA with methylation-sensitive restriction enzymes SacII () and SmaI (). DNA was digested with the indicated enzymes and analyzed by Southern Blot hybridization with p123. The probe identifies restriction fragments from the 5′end of the RB1 gene and a 28S rDNA fragment. This figure shows the results of digesting DNA of 7 tumors with SacI and SacII respectively SacI and SmaI. The osteosarcoma DNA show fragments as expected if SacII respectively SmaI restriction sites are not methylated. Kb = kilobases. (a) 1 = Placenta DNA as normal control only with SacI; 2 = placenta DNA with SacI and SacII; 3–9 = osteosarcoma DNA with SacI and SacII. (b) 1 = Placenta DNA only with SacI; 2 = placenta DNA with SacI and SmaI; 3–10 = osteosarcoma DNA with SacI and SmaI.
3 Hypermethylated RB1 gene derived from a sporadic retinoblastoma as positive control. 1 = Hypermethylated RB1 gene derived from a sporadic retinoblastoma digested with SacI and SacII. The 6.1-kb fragment persists indicating methylated restriction sites. 2 = Unmethylated RB1 gene derived from an osteosarcoma digested with SacI and SacII. 3 = Hypermethylated RB1 gene derived from a sporadic retinoblastoma digested with SacI and SmaI. 6.1-kb, 4.0-kb, and 2.0-kb fragments due to heterogenous methylation (Greger et al. 1989). 4 = Unmethylated RB1 gene derived from an osteosarcoma digested with SacI and SmaI.
