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Nucleic Acids Research Advance Access originally published online on November 12, 2007
Nucleic Acids Research 2007 35(21):e144; doi:10.1093/nar/gkm984
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Nucleic Acids Research, 2007, Vol. 35, No. 21 e144
© 2007 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

A ligation assay for multiplex analysis of CpG methylation using bisulfite-treated DNA

Christina Dahl and Per Guldberg*

Department of Genomics and Danish Centre for Translational Breast Cancer Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark

*To whom correspondence should be addressed. Tel: +45 3525 7500; Fax: +45 3525 7721; Email: perg{at}cancer.dk

Received July 11, 2007. Revised September 28, 2007. Accepted October 20, 2007.

Aberrant methylation of promoter CpG islands is causally linked with a number of inherited syndromes and most sporadic cancers, and may provide valuable diagnostic and prognostic biomarkers. In this report, we describe an approach to simultaneous analysis of multiple CpG islands, where methylation-specific oligonucleotide probes are joined by ligation and subsequently amplified by polymerase chain reaction (PCR) when hybridized in juxtaposition on bisulfite-treated DNA. Specificity of the ligation reaction is achieved by (i) using probes containing CpGpCpG (for methylated sequences) or CpApCpA (for unmethylated sequences) at the 3' ends, (ii) including three or more probes for each target, and (iii) using a thermostable DNA ligase. The external probes carry universal tails to allow amplification of multiple ligation products using a common primer pair. As proof-of-principle applications, we established duplex assays to examine the FMR1 promoter in individuals with fragile-X syndrome and the SNRPN promoter in individuals with Prader-Willi syndrome or Angelman syndrome, and a multiplex assay to simultaneously detect hypermethylation of seven genes (ID4, APC, RASSF1A, CDH1, ESR1, HIN1 and TWIST1) in breast cancer cell lines and tissues. These data show that ligation of oligonucleotide probes hybridized to bisulfite-treated DNA is a simple and cost-effective approach to analysis of CpG methylation.


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L. S. Kristensen, T. Mikeska, M. Krypuy, and A. Dobrovic
Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection
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[Abstract] [Full Text] [PDF]


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