Published online 15 March 2008 Nucleic Acids Research, 2008, Vol. 36, No. 7 e42 doi:10.1093/nar/gkn113 Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high- throughput and probe-free quantitative DNA methylation detection Lasse S. Kristensen1,2, Thomas Mikeska 1 , Michael Krypuy1 and Alexander Dobrovic1,3,* 1Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1 A’Beckett Street, Melbourne, Victoria 8006, Australia, 2Institute of Human Genetics, University of Aarhus, The Bartholin Building, DK-8000 Aarhus C, Denmark and 3Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia Received December 19, 2007; Revised January 24, 2008; Accepted February 19, 2008 ABSTRACT DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely quali- tative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorpo- rates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methyla- tion. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP. INTRODUCTION In mammalian cells, DNA methylation occurs almost exclusively at the carbon-5 position of cytosine residues within CpG dinucleotides. The CpG dinucleotide is distributed in a non-random fashion throughout the human genome. CpG-depleted regions are interspersed with CpG-rich sequences referred to as CpG islands (1). These islands are often located at promoter regions of protein encoding genes and tend to be unmethylated (2,3). Aberrant DNA methylation patterns are one of the hallmarks of cancer. In most cancers, promoter hyper- methylation correlates with gene silencing. This has been shown for a wide range of tumour suppressor genes including the genes studied here and reviewed in (2,3): the cell-cycle inhibitor gene CDKN2A (p16INK4a), the pro- apoptotic death-associated protein kinase gene DAPK1, the cell-adhesion gene CDH1 and the retinoic acid receptor gene RARB. In cancer, methylation of some promoter CpG islands can be an early event, and thus the detection of methyl- ation shows great promise as a biomarker for early detection (4–6). Conventional methods for cancer detec- tion are in general not capable of ﬁnding pre-neoplastic and small malignant lesions, and are thus not suitable for early detection. Molecular biomarkers in body liquids such as blood, sputum or urine that allow detection and diagnosis of tumours at an early stage would be ideal. However, in these types of samples, tumour-derived material is hard to detect because of the presence of material from normal cells, and thus highly sensitive methods are needed (7). As one example, methylation of the CDKN2A promoter has been detected in the sputum of smokers up to 3 years before they are diagnosed with cancer (8). Detection of low level methylation also shows great potential in the molecular monitoring of established disease after therapy (4). This has already been shown to be feasible in various cancers using DNA derived from plasma or serum (9,10). *To whom correspondence should be addressed. Tel: +61 3 9656 1807; Fax: +61 3 9656 1460; Email: alexander.dobrovic@petermac.org 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.