PAGE 5 OF 13 Nucleic Acids Research, 2008, Vol. 36, No. 7 e42 Figure 2. Melting proﬁles of a true positive result for each SMART-MSP assay. Universally methylated template was ampliﬁed and analysed by HRM analysis. Each assay has a characteristic melting proﬁle. (A) The CDH1 assay. (B) The DAPK1 assay. (C) The CDKN2A assay. (D) The RARB assay. between the primers, the HRM step can function as a control for ampliﬁcation of incompletely converted DNA. If incomplete converted sequences are ampliﬁed, the amplicon will have a higher GC-content relative to fully converted amplicons, and will therefore melt later. We designed the DAPK1 and CDKN2A assays in this way. Second, when CpG sites are included between the primers, the HRM step can be used to assess if these CpG sites are methylated. We designed the CDH1 and RARB assays in this way. Generally, if left-shifted melting is observed, this is an indicator that some or all of the CpG sites are not methylated. A complex melting pattern consisting of heteroduplexes as well as homoduplexes can occur if more than one molecule is ampliﬁed during the PCR and the studied region is heterogeneously methy- lated. Thus, a heterogeneously methylated region can give a melting proﬁle extending to the left, due to the melting of molecules with diﬀerent CpG positions being methyl- ated and heteroduplex formation between them (Figure 1). Left shifting can also indicate false positives due to false priming. In this case, a single left-shifted peak will be seen. Generally, false priming is associated with very late ampliﬁcation (Figure 1). False priming can generally be minimized by stringent PCR conditions. Assays should be designed to include as few non-CpG cytosines as possible, preferably none. A conversion control should be per- formed in parallel when non-CpG cytosines cannot be avoided to be conﬁdent that these are converted. Our CDH1 and RARB primers were designed so that they can also be used with a MethyLight probe for validation of the SMART-MSP methodology. Because of this, more non-CpG cytosines are found in between the primers than what would be ideally preferred. Generally, longer products give more intrinsic variation of the melting curves (unpublished results). Third, small amplicons can be designed that do not include non-CpG cytosines or CpG sites. In this case, only the methylation status of those CpG sites found under the primers are assessed, and the HRM step is used only then to validate that the correct sequence is ampliﬁed. In all the above cases, the presence of primer dimers and non-speciﬁc products can be identiﬁed on the basis of aberrant melting proﬁles by HRM analysis (data not shown) without the need for gel electrophoresis. Melting profiles of the SMART-MSP assays The melting proﬁles of a true positive result, obtained by amplifying standards containing methylated template for each assay, were used as references for unknown samples (Figure 2). Since all the ampliﬁed dilution standards for a given amplicon have the same melting proﬁle, we could have chosen any one of them as a reference. The melting temperature of each assay was approximately 83.28C for CDH1, 79.58C for DAPK1, 75.88C for CDKN2A and 80.88C for RARB. Conﬁrmatory gel electrophoresis was performed in the development phase for all assays, and only one band of the expected size was observed (data not shown).