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Nucleic Acids Research 2005 33(10):e93; doi:10.1093/nar/gni092
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Published online 8 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

MSRE-PCR for analysis of gene-specific DNA methylation

Anatoliy A. Melnikov1, Ronald B. Gartenhaus1,2, Anait S. Levenson1,3, Natalia A. Motchoulskaia1 and Victor V. Levenson (Chernokhvostov)1,*

1Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University Chicago, IL 60611, USA 2Division of Hematology/Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University Chicago, IL 60611, USA 3Department of Orthopaedic Surgery, Feinberg School of Medicine, Northwestern University Chicago, IL 60611, USA

*To whom correspondence should be addressed. Tel: +1 312 503 2435; Fax: +1 312 503 3063; Email: levenson{at}northwestern.edu

Received January 25, 2005. Revised April 18, 2005. Accepted May 18, 2005.

Abnormal DNA methylation is observed in certain promoters of neoplastic cells, although the likelihood of methylation for each individual promoter varies. Simultaneous analysis of many promoters in the same sample can allow use of statistical methods for identification of neoplasia. Here we describe an assay for such analysis, based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers (MSRE-PCR). MSRE-PCR includes extensive digestion of genomic DNA (uncut fragments cannot be identified by PCR), can be applied to dilute samples (<1 pg/µl), requires limited amount of starting material (42 pg or genomic equivalent of seven cells) and can identify methylation in a heterogeneous mix containing <2% of cells with methylated fragments. When applied to 53 promoters of breast cancer cell lines MCF-7, MDA-MB-231 and T47D, MSRE-PCR correctly identified the methylation status of genes analyzed by other techniques. For selected genes results of MSRE-PCR were confirmed by methylation-specific PCR and bisulfite sequencing. The assay can be configured for any number of desired targets in any user-defined set of genes.

Present address: Ronald B. Gartenhaus, University of Maryland, Greenebaum Cancer Center, 9-011 BRB, 655 West Baltimore Street, Baltimore, Maryland 21201, USA


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