e42 Nucleic Acids Research, 2008, Vol. 36, No. 7 selective for methylated template. However, this also facilitates ampliﬁcation of incomplete converted sequences in the bisulphite-treated DNA as unconverted sequence resembles methylated sequence. It is possible that only a small subset of the DNA copies suﬀer a substantially lower conversion rate, which in combination with the high sensitivity of MSP can lead to false positives or over- estimation of results. Also, it is possible that the distribution of unconverted sites is non-random, thus making some promoter regions more prone to incomplete conversion than others, and that conversion rates are dependent on DNA quality (16). Thus, in spite of stringent PCR conditions, a false-positive result can arise if bisulphite conversion is incomplete. For these reasons, it is recommended that a control for incomplete conversion is performed. This control should preferably assess the conversion status of non-CpG cytosines in the vicinity of the primer positions. We have shown that having a non-CpG cytosine at the 30-end is not suﬃcient to rule out the possible ampliﬁcation of incomplete converted DNA (Figures 5 and 6). If the annealing temperature is too low or too many cycles are used, ampliﬁcation can occur across the 30 mismatch. This type of false positive can be detected by the use of an appropriate negative control (e.g. WGA product). Finally, the sensitivity of MSP might lead to false positives because of the ampliﬁcation of a rare subpopula- tion of methylated sequences. The tumour sample might be extremely heterogeneous, with only a small proportion of methylated cells. Alternatively, the methylation might derive from normal cells in the tumour biopsy (12,33). In either case, it would be incorrect to call the tumour methylated for that particular gene. This problem arises because MSP is a non-quantitative methodology. Despite these limitations, MSP remains useful, particularly in preliminary screening of a large number of tumour specimens but may not be suitable in a clinical setting (7,34). Here, we have shown that SMART-MSP can give accurate quantitative data for DNA methylation detec- tion. The combination of MSP with HRM, which is enabled by the use of a HRM-compatible DNA double- stranded intercalating dye, enables the sensitive screening of the region in between the MSP primers. Thus, infor- mation is provided that cannot be obtained by electro- phoresis. For full utility of the methodology, one or both of two types of primer positioning should be used: (i) only non-CpG cytosines between the primers allowing assess- ment if (low) levels of ampliﬁcation are due to incomplete conversion and (ii) CpGs (with as few non-CpG cytosines as possible) between the primers allowing assessment if (low) levels of ampliﬁcation are due to partial or heterogeneous methylation. Interpretation is made by considering both the quantiﬁcation and the HRM information (Figure 1). When the melt proﬁle of a true positive is established (Figure 2) there is no need for gel electrophoresis analysis or any further processing, and thus SMART-MSP is a closed-tube method. HRM analysis can easily detect single base pair changes in the ampliﬁed DNA sequence. Thus, by including PAGE 10 OF 13 non-CpG cytosines between the primers, the conversion status of these can be assessed. If cytosines in between the primers are not converted, the amplicons will melt late relative to amplicons derived from fully converted template (Figure 1). By generating incompletely converted DNA sequences, and using these as templates for our CDKN2A and DAPK1 assays, we were able to show that ampliﬁcation from these usually occurred, and were easily identiﬁed as they always corresponded to right-shifted melting peaks (Figure 5). In addition, we ampliﬁed unmodiﬁed DNA as well as modiﬁed DNA derived from the same individual using primers that select poorly between modiﬁed and unmodiﬁed DNA, and observed highly reproducible right-shifted melting peaks from the unmodiﬁed template relative to the modiﬁed one (Figure 6). False-positive results due to false priming can be detected by HRM analysis as well, if CpGs are included in between the primers (Figure 1). By running the CDH1 SMART-MSP assay for an additional 10 cycles, late ampliﬁcation from the fully unmethylated control (WGA product) was seen. In this case, a left-shifted melting peak was observed due to the two CpGs found in between the primers being unmethylated. This allowed us to readily identify it as a false-positive result (Figure 7). However, suﬃciently stringent PCR conditions and adequate primer design are the best insurance against false priming. A left-shifted peak can also be due to the target sequence being heterogeneously methylated, which can result in heteroduplex formation, and thus the melting proﬁle will often be visually diﬀerent (Figure 1). The exact number of CpG sites that need to be methylated within MSP primers under given PCR conditions is hard to assess. This is especially important to keep in mind when heterogeneously methylated sequences are studied. Thus, methodologies that utilize MSP primers are only semi- quantitative when heterogeneously methylated DNA is ampliﬁed. For this reason, MSP might be less suited in those cases when CpG islands show highly variable methylation, as has been reported for the p15INK4B and CX26 (connexin 26) genes (35–37). However, when including CpG sites in between the primers in the SMART-MSP methodology, it can be assessed whether the studied region is heterogeneously methylated or not, by the HRM analysis. We were able to reliably detect the 0.1% methylated standard for each assay with high reproducibility using 25 ng of bisulphite-modiﬁed DNA as template (Figure 3). Ampliﬁcation from the 0.01% methylated standard was occasionally observed in all SMART-MSP assays, but generally showed high run to run variations in CT values. This might, in part, be caused by very low methylation levels present in normal blood. However, the quantitative accuracy in the range from 100% methylated template to 0.1% methylated template was not impaired by the fact that we did the dilutions in DNA from normal blood. During WGA of DNA all detectable methylation infor- mation is lost, and thus it is possible to create fully unmethylated template. However, results can be biased during normalization for DNA input if the dilutions are done using WGA product, since diﬀerent genes are not