Figure 2

Verification of tumor-specific methylation of seven candidate target genes. These targets were identified by the MIRA-assisted microarray approach (target numbers 5, 7, 8, 29, 40, 42 and 54, corresponding to TLX1, CNTNAP1, GFI1, MT1E, NR2E1, CPEB1 and HLXB9, respectively; Table 1). Genomic DNA from ductal carcinomas in situ (T) and matching normal breast tissues (N) was treated with sodium bisulfite and the target CpG island sequences were amplified using gene-specific primers. Methylation was confirmed by a BstUI combined bisulfite restriction analysis assay, which produces digestion products when BstUI restriction sites are methylated and not converted by bisulfite. HeLa DNA was methylated in vitro with the SssI methyltransferase and served as a positive control (Meth. DNA, Methylated HeLa DNA). Vertical white arrows indicate hypermethylated alleles in the target sequence. ±, digestion was carried out with or without BstUI restriction enzyme; N/T, normal/tumor pairs. When matching normal tissue was not available, a DNA mixture derived from several normal breast tissues was used instead. M, DNA marker.