e42 Nucleic Acids Research, 2008, Vol. 36, No. 7 Methylation-speciﬁc PCR (MSP) (11) is a highly sensi- tive method for the detection of low level methylation, and can be sensitive to at least 0.1% methylated template. However, MSP is prone to false-positive results (12–14). MSP primers are normally designed to have one or more cytosines of CpG sites at or near the 30 end. This makes the primers highly selective for methylated template, but also facilitates ampliﬁcation of incompletely converted sequences in the bisulphite-treated DNA (13). It is thought that bisulphite treatment, in spite of recent improvements in this area, still remains the main source of variability in the analysis of DNA methylation. Recent results show that incomplete conversion may typically be in the order of 2%, even when a commercial kit is used (15,16). This variability can not only lead to false-positive results, but can also impair quantitative assays in a way that leads to overestimation of methylation levels (17), especially when looking at low level methylation. Diﬀerent methods for the detection of incompletely converted products co-ampliﬁed during the PCR have been proposed (14,16,18). These methods are relatively labour intensive and require removal of the PCR product from the tube for further analysis creating the potential for PCR contamination, or the use of additional probes as in the ConLight-MSP methodology (14). MSP has been made quantitative by the use of ﬂuorescent TaqMan probes enabling real time detection of MSP products (such as in the MethyLight technique) (19–21). This also eliminates any signal from non-speciﬁc ampliﬁcation. However, the introduction of a probe com- plicates assay design, and can result in heterogeneously methylated sequences that would otherwise be detected by MSP being missed, because of the need for the probe to hybridize correctly before a signal is observed. Here we present a new method: Sensitive Melting Analysis after Real Time (SMART)-MSP for sensitive DNA methylation detection based on probe-free real-time PCR. With the use of a new generation of ﬂuorescent dyes, which do not inhibit PCR when they intercalate into double-stranded DNA at saturating levels (22), highly accurate quantiﬁcation and further analysis of the amplicon by high-resolution melting (HRM), has become possible. As the temperature increases during the HRM step, and the DNA ‘melts’, the dye is released and the signal of ﬂuorescence decreases rapidly. This change in ﬂuorescence is sequence speciﬁc and can be monitored by appropriately designed instrumentation. SMART-MSP provides information that cannot be obtained by electrophoresis, and thus functions as a quality control to avoid false-positive results caused by incomplete conversion or false priming due to less stringent PCR conditions. Primer dimers and non-speciﬁc products can be detected as well. We developed SMART-MSP assays for several tumour suppressor genes that are currently used as molecular biomarkers for early cancer detection or are likely to have a high potential as such. The quantitative accuracy of the assays was tested using a standard dilution series of methylated DNA into unmethylated DNA. Furthermore, we show that false-positive results due to incomplete bisulphite conversion or false priming can be identiﬁed by PAGE 2 OF 13 HRM analysis and we have analysed a panel of cell lines, and breast cancer samples by the SMART-MSP methodology. MATERIALS AND METHODS Samples and DNA extraction The investigations were performed after approval by the Peter MacCallum Cancer Centre Ethics of Human Research committee (Projects 02/70 and 02/26). Puriﬁed genomic DNA from cell lines (2008, MCF7, HS578T, MCF10A, MDA-MB-468, MDA-MB-231, MDA-MB- 435, PC3, SKBr-3, Colo205, RPMI8226, SW948, HL-60 and T47D) was available in our laboratory. The DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufac- turer’s instructions. Breast cancer samples were provided by the Peter MacCallum Cancer Centre Tissue Bank. DNA from those samples was extracted using the salting out method (23). Universal Methylated DNA (Chemicon, Millipore, Billerica, MA, USA) was used as a fully methylated positive control. DNA from peripheral blood mononuclear cells from normal individuals was used as unmethylated DNA for dilutions. Standard dilution series of 100, 10, 1, 0.1 and 0.01% methylation levels were prepared by diluting the fully methylated DNA into the unmethylated DNA. DNA was quantiﬁed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Whole-genome amplification Whole-genome ampliﬁcation (WGA) was performed as described by Umetani et al. (24) with slight modiﬁcations. Brieﬂy, for primary WGA, 1 ng of genomic DNA extrac- ted from peripheral blood mononuclear cells in 1 ml was subjected to ampliﬁcation (2 h at 308C) with the Illustra GenomiPhi V2 DNA Ampliﬁcation Kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. For secondary WGA, 0.1 ml of the unpuriﬁed primary WGA product was diluted in water to a ﬁnal volume of 1 ml and processed as described above. The secondary WGA product was puriﬁed using the QIAquick PCR Puriﬁcation Kit (Qiagen) according to the manufacturer’s instructions, eluted in 50 ml buﬀer EB, and quantiﬁed with a NanoDrop ND-1000 spectro- photometer. The secondary WGA product was used as fully unmethylated DNA (negative control). Bisulphite modification Five hundred nanograms of genomic DNA or WGA product was subjected to bisulphite conversion with the EpiTect Bisulﬁte kit (Qiagen) according to the manu- facturer’s instructions. The puriﬁed, bisulphite-modiﬁed DNA (WGA product) was eluted two times in 20 ml buﬀer EB. Incomplete bisulphite-modiﬁed DNA was prepared by treating the appropriate samples for 20 or 40 min with the bisulphite mix solution and puriﬁed as described earlier.