Figure 3

Methylation analysis of the human DKK3 promoter. (A) A 2.0 kb genomic sequence of the DKK3 promoter, analyzed by methprimer software [29], revealed the presence of two CpG islands (blue); one between relative position 834 and 1261 and another one between position 1529 and 1917. Two alternative tissue-specific DKK3 transcripts have been described [30]. Since transcription of the shorter transcript is widely distributed in normal tissues, we chose the region of the second transcription start site (TSS, arrow) for methylation analysis. Position 1000 indicates the alternative transcription start site of the longer transcript (TSS*, arrow). A region of high CpG (red vertical bars) densitiy within the second CpG island was chosen for MSP analysis. (B) Sensitivity of the utilized MSP primers was determined by a dilution series of methylated DNA with unmethylated DNA (Epi Tect control DNA, Qiagen). At least 1% of methylated DNA (~0.1 ng) can be detected with the DKK3 MSP primers. bp, base pair marker; NTC, 'no template control'.