PAGE 11 OF 13 ampliﬁed in the exact same proportions. Nevertheless, WGA product is excellent as a negative control, especially when looking at low level methylation. MSP was originally made quantitative by the use of TaqMan probes (19–21), but quantiﬁcation without probes using the dye SYBR Green has been reported (38–41). SYBR Green intercalation into double-stranded DNA has been shown to be markedly inﬂuenced by salt concentration, by dye/base pair ratios which are not constant during the PCR since more and more double- stranded DNA is generated through each cycle, and to show sequence speciﬁc binding (42). For this reason, the number of PCR cycles can inﬂuence melting curve analysis (43). Generally, some of the problems associated with SYBR Green can be minimized if high dye/base pair ratios are used. However, SYBR Green cannot be used at saturating concentrations without inhibiting the PCR. These problems have been markedly reduced by the introduction of a new generation of dyes (22). It has previously been shown that melting analysis can discriminate methylated from unmethylated DNA (44,45). These assays were based on methylation-independent PCR (MIP) primers, and have not become widely used, presumably because of the technical limitations of reagents, instrumentation and data analysis software used at that time. Generally, methods utilizing MIP primers can be compromised by the PCR bias phenom- enon (46), but this is not an issue when MSP primers are used. Melting curve analysis has also been used in combina- tion with the MSP methodology as an alternative to gel electrophoresis (47). This methodology did not provide quantitative data or information that cannot be provided by gel electrophoresis. Melting analysis of MSP products in the presence of SYBR green have also been used to detect primer dimers (38). This can be done by gel electrophoresis as well. This study did provide quantita- tive data, but these were much less accurate compared to what we have obtained with a dye that does not inhibit PCR. SMART-MSP is complementary to our previously described methodology using HRM, methylation-sensitive HRM (MS-HRM) (48). SMART-MSP uses MSP primers and quantiﬁcation is based on CT values instead of melting curve comparisons. Also SMART-MSP can detect the ampliﬁcation of incompletely converted DNA, and is generally more sensitive. However, the main advantage of SMART-MSP might be that each assay is performed at one annealing temperature; where as in MS-HRM, a range of diﬀerent temperatures are needed for the sensitive screening of samples showing markedly diﬀerent methyla- tion levels. In MS-HRM, quantiﬁcation is based on comparisons with melting proﬁles of a standard dilution series that needs to be included in every run. This is not necessary when performing SMART-MSP assays which quantify relative to a 100% methylated control and the ampliﬁcation of a CpG-free control sequence. We are currently using MS-HRM to analyse samples where relatively high levels of methylation are expected whereas SMART-MSP comes into its own to detect low levels of methylation. Nucleic Acids Research, 2008, Vol. 36, No. 7 e42 Compared to the MethyLight technology, SMART- MSP does not require expensive probes. However, quantiﬁcation without probes is compromised by primer dimers and non-speciﬁc ampliﬁcation (38). For this reason, there is a need for evaluation of the PCR product, which can be conveniently done with HRM analysis. We observed non-speciﬁc ampliﬁcation from some of our controls when the assays were performed at lower annealing temperatures. These products melted diﬀer- ently, and could be identiﬁed as non-speciﬁc ampliﬁcation using gel electrophoresis as well (data not shown). However, when the assays were performed at the optimized annealing temperature, no primer dimers or non-speciﬁc products were observed. Without probes, less optimization may be needed and assay design has become easier. The use of HRM can give information about the methylation status of CpGs between the primers, but most importantly, the HRM step can be used as a control to indicate ampliﬁcation of incompletely modiﬁed sequences, false priming or non-speciﬁc products. Thus, SMART- MSP is less prone to false-positive results and over- estimation of methylation levels. We have shown that the sensitivity of our assays is similar to what has been reported for MethyLight. In conclusion, SMART-MSP has made quantitative MSP inexpensive, more accurate, and less prone to false positives. It is a closed-tube method based on a high- throughput methodology, and thus it might prove to be the method of choice for the assessment of DNA methyl- ation in clinical samples, particularly when low levels of methylation need to be sensitively and accurately determined. ACKNOWLEDGEMENTS We acknowledge Chelsee Hewitt and Angela Tan for critically reading this manuscript, and Stephen Fox and David Westerman for their continued support. This work was funded by grants from the United States Department of Defense Breast Cancer Research Program (BC046207) and the National Health and the Medical Research Council of Australia (350452) to A.D. Funding to pay the Open Access publication charges for this article was provided by Department of Defense (US) Breast Cancer Research Program. Conﬂict of interest statement. A provisional patent has been ﬁled for this methodology by the Peter MacCallum Cancer Centre. REFERENCES 1. Gardiner-Garden,M. and Frommer,M. (1987) CpG islands in vertebrate genomes. J. Mol. Biol., 196, 261–282. 2. Esteller,M. (2005) Aberrant DNA methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicol., 45, 629–656. 3. Herman,J.G. and Baylin,S.B. (2003) Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med., 349, 2042–2054. 4. Laird,P.W. (2003) The power and the promise of DNA methylation markers. Nat. Rev. Cancer, 3, 253–266.