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Nucleic Acids Research Advance Access published online on March 15, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn113
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© 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.

Methods Online

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 Mikeska1, 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

*To whom correspondence should be addressed. Tel: +61 3 9656 1807; Fax: +61 3 9656 1460; Email: alexander.dobrovic{at}petermac.org

Received December 19, 2007. Revised January 24, 2008. Accepted February 19, 2008.

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 qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates 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 methylation. 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.


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