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Nucleic Acids Research 2004 32(21):e168; doi:10.1093/nar/gnh166
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Published online 2 December 2004

Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved

MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome

Hélène Thomassin, Clémence Kress and Thierry Grange*

Institut Jacques Monod du CNRS, Universités Paris 6-7, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France

* To whom correspondence should be addressed. Tel: +33 1 44275707; Fax: +33 1 44275716; Email: grange{at}ccr.jussieu.fr
Present address: Hélène Thomassin, Institut Pasteur, CNRS URA 2578, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France

Received September 28, 2004; Revised and Accepted November 5, 2004

Here we present MethylQuant, a novel method that allows accurate quantification of the methylation level of a specific cytosine within a complex genome. This method relies on the well-established treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil without modifying 5-methyl cytosine. The region of interest is then PCR-amplified and quantification of the methylation status of a specific cytosine is performed by methylation-specific real-time PCR with SYBR Green I using one of the primers whose 3' end discriminates between the methylation states of this cytosine. The presence of a locked nucleic acid at the 3' end of the discriminative primer provides the specificity necessary for accurate and sensitive quantification, even when one of the methylation states is present at a level as low as 1% of the overall population. We demonstrate that accurate quantification of the methylation status of specific cytosines can be achieved in biological samples. The method is high-throughput, cost-effective, relatively simple and does not require any specific equipment other than a real-time PCR instrument.


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