e42 Nucleic Acids Research, 2008, Vol. 36, No. 7 PAGE 8 OF 13 Figure 5. Validation of the conversion control in the DAPK1 and CDKN2A assays. A peripheral blood control sample was bisulphite treated using diﬀerent times of conversion (20 min, 40 min, normal protocol), and used to test the conversion control of these assays. (A) The DAPK1 assay. A gradual right-shift of the melting peaks was observed as the treatment time decreases. The observed right-shift of the incompletely treated samples indicates that some of the non-CpG cytosines in between the primers were not converted. Thus, these samples could be identiﬁed as false positives. (B) The CDKN2A assay. The 40 min treated sample and the 20 min treated sample both showed right-shifted melting peaks. Again, indicating that some of non-CpG cytosines in between the primers were not converted, and thus these samples could also be identiﬁed as false positives. shown. These results were validated using the RARB MethyLight assay, in which the same six samples were shown to be methylated at similar levels (Figure 8D). These ﬁndings are in agreement with what has recently been reported for this locus in breast cancer (31,32). None of the samples showed high-level methylation at the CDH1 promoter. We found 11 to be methylated between 0.1% and 0.7% which could be detected with high reproducibility. Furthermore, we found 10 samples to be methylated in the interval from 0.01% to 0.1%, for which the reproducibility between samples was less good. These observed values might only account for a biologi- cally insigniﬁcant background methylation level that can be found in normal cells (unpublished results). A recent study has found that a low background of CDH1 methylation is present in normal breast tissue, and that this level did not diﬀer signiﬁcantly from what was found in adjacent malignant breast tissue (32). Another recent study reports that CDH1 methylation was not found in a cohort of 28 tissue biopsies from 17 breast Figure 6. Detection of false priming from a whole genome ampliﬁed template. We used an assay that selected poorly against unmodiﬁed templates. In this assay, ﬁve non-CpG cytosines and no CpG sites are found in between the primers. These non-CpG cytosines were converted to uracil in the bisulphite-modiﬁed template (red), but not in the unmodiﬁed template (blue). Thus, a signiﬁcant right-shift of the melting proﬁle of the unmodiﬁed amplicon is observed. (A) Real-time PCR ampliﬁcation data. (B) First derivative melting peaks. cancer patients (31). However, the method used in that study was not sensitive enough to detect methylation levels 1%. Thus, if CDH1 methylation is detected using a purely qualitative and highly sensitive methodol- ogy, positive results might arise due to low background methylation. We tested the same samples using our CDH1 MethyLight assay with comparable results (data not shown). However, the CDH1 MethyLight assay was not able to detect methylation in four of the samples showing the lowest levels of methylation in the CDH1 SMART- MSP assay. DISCUSSION MSP is a widely used method for the detection of DNA methylation. It uses primers speciﬁc for methylated (and optionally unmethylated), bisulphite-modiﬁed DNA (11). MSP is based on the principle that primers with mis- matched 30-ends will not be capable of extension during the PCR. If a band that corresponds to the amplicon size given by the MSP primers is seen on a gel after PCR, it is concluded that the sample is methylated. MSP is possibly