p15 promoter hypermethylation. (A) Schematic representation of p15 exon 1 and surrounding HindIII (H3) and EagI (E) sites. The p15 probe used (indicated above) hybridizes to exon 1. A 2.8-kb band is seen when the samples are digested with HindIII alone. When the samples are digested withEagI (a methylation-sensitive enzyme) in combination withHindIII, a 0.6-kb band is recognized by the probe if the gene is unmethylated (cuts at EagI) and a 2.8-kb band is present if the sample is methylated (does not cut at EagI) (note that a 2.2-kb band is not seen, because the exon 1 probe does not hybridize to this region of DNA). (B) Autoradiogram of a blot containing HindIII and EagI-in combination with HindIII-digested DNAs hybridized with the p15 exon 1 probe. The locations of the expected 2.8-kb and 0.6-kb bands are indicated by arrows at the left. Samples include DNA from Kg1A cell line, a healthy control (NORM), DR#10 at diagnosis (Dx) and at relapse (R). Homozygous methylation is seen in Kg1A, and hemizygous methylation in DR#10 at relapse, but not at initial diagnosis.