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Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA)

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DNA Methylation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1708))

Abstract

This chapter describes a method for the rapid assessment of promoter hypermethylation levels or methylation of imprinted regions in human genomic DNA extracted from various sources using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA).

Multiplex ligation-dependent probe amplification (MLPA) is a powerful and easy-to-perform PCR-based technique that can identify gains, amplifications, losses, deletions, methylation and mutations of up to 55 targets in a single reaction, while requiring only minute quantities of DNA (about 50 ng) extracted from blood, fresh frozen or formalin-fixed paraffin-embedded materials. Methylation-specific MLPA (MS-MLPA) is a variant of MLPA, which does not require sodium bisulfite conversion of unmethylated cytosine residues, but instead makes use of the methylation-sensitive endonuclease HhaI. MS-MLPA probes are designed to contain a HhaI recognition site (GCGC) and thus target one CpG dinucleotide within a CpG island. If the HhaI recognition site is not methylated, HhaI will cut the probe–sample DNA hybrid and no PCR product will be formed. If the target DNA is methylated, HhaI is not able to cut, and the fragment will be amplified during subsequent PCR. For data analysis, MS-MLPA peak patterns of the HhaI-treated and -untreated reactions are compared, leading to calculation of a methylation percentage. The methylation profile of a test sample is assessed by comparing the probe methylation percentages obtained on the test sample to the percentages of the reference samples. MS-MLPA can be combined with copy number and point mutation detection in the same reaction.

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Authors and Affiliations

  1. Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, PO Box 85500, Utrecht, 3508 GA, The Netherlands

    Cathy B. Moelans & Paul J. van Diest

  2. MRC-Holland, Willem Schoutenstraat 1, Amsterdam, 1057 DL, The Netherlands

    Lilit Atanesyan & Suvi P. Savola

Authors
  1. Cathy B. Moelans
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  2. Lilit Atanesyan
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  3. Suvi P. Savola
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  4. Paul J. van Diest
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Corresponding author

Correspondence to Cathy B. Moelans .

Editor information

Editors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

    Jörg Tost

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Moelans, C.B., Atanesyan, L., Savola, S.P., van Diest, P.J. (2018). Methylation-Specific Multiplex Ligation-Dependent Probe Amplification (MS-MLPA). In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_27

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  • DOI: https://doi.org/10.1007/978-1-4939-7481-8_27

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7479-5

  • Online ISBN: 978-1-4939-7481-8

  • eBook Packages: Springer Protocols

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