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Nucleic Acids Research 2005 33(14):e128; doi:10.1093/nar/gni127
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Published online 16 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences

Anders O. H. Nygren1,2, Najim Ameziane2, Helena M. B. Duarte1, Raymon N. C. P. Vijzelaar1, Quinten Waisfisz3, Corine J. Hess3, Jan P. Schouten1 and Abdellatif Errami1,*

1MRC-Holland bv Amsterdam, The Netherlands 2Department of Clinical and Human Genetics, VU University Medical Center Amsterdam, The Netherlands 3Department of Hematology, VU University Medical Center Amsterdam, The Netherlands

*To whom correspondence should be addressed. Tel: +31 20 4891248; Fax: +31 20 6891149; Email: a.errami{at}vumc.nl

Received May 20, 2005. Revised June 23, 2005. Accepted July 25, 2005.

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.


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