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Mol Genet Genomics. 2011 Oct;286(3-4):279-91. doi: 10.1007/s00438-011-0645-9. Epub 2011 Aug 31.

Methylation-specific ligation detection reaction (msLDR): a new approach for multiplex evaluation of methylation patterns.

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1
Institute of Pathology, University Medicine Charité, Charitéplatz1, 10117 Berlin, Germany. felix.bormann@charite.de

Abstract

A new sensitive method for multiplex gene-specific methylation analysis was developed using a ligation-based approach combined with a TaqMan-based detection and readout employing universal reporter probes. The approach, termed methylation-specific Ligation Detection Reaction (msLDR), was applied to test 16 loci in 8 different colorectal cancer cells in parallel. These loci encode immune regulatory genes involved in T-cell and natural killer cell activation, whose silencing is associated with the development or progression of colorectal cancer. Parallel analysis of HLA-A, HLA-B, STAT1, B2M, LMP2, LMP7, PA28α, TAP1, TAP2, TAPBP, ULBP2 and ULBP3 by msLDR in eight colorectal cancer cell lines showed preferential methylation at the HLA-B, ULBP2 and ULBB3 loci, but not at the other loci. MsLDR was found to represent a suitable and sensitive method for the detection of distinct methylation patterns as validated by conventional bisulphite Sanger sequencing and COBRA analysis. Since gene silencing by epigenetic mechanisms plays a central role during transformation of a normal differentiated somatic cell into a cancer cell, characterization of the gene methylation status in tumours is a major topic not only in basic research, but also in clinical diagnostics. Due to a very simple workflow, msLDR is likely to be applicable to clinical samples and thus comprises a potential diagnostic tool for clinical purposes.

PMID:
21879293
DOI:
10.1007/s00438-011-0645-9
[Indexed for MEDLINE]
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