PCR Protocols pp 193-205 | Cite as

The Many Faces of MLPA

  • Thomas Ohnesorg
  • Erin Turbitt
  • Stefan J. White
Part of the Methods in Molecular Biology book series (MIMB, volume 687)


Multiplex Ligation-dependent Probe Amplification (MLPA) is a PCR-based technique that was developed for identifying deletions and duplications in genomic DNA. The simplicity and sensitivity of this approach has led to it being implemented in many laboratories around the world. Since the original publication, there have been several variants of MLPA described, allowing the quantitative analysis of mRNA transcript levels, CpG methylation, complex genomic regions, and DNaseI hypersensitive sites. This chapter outlines the basic MLPA protocol, describes the different modifications and applications that have been published, and discusses the critical points during each of the steps.

Key words

MLPA Copy number variation Deletion Duplication Mutation screening Methyla­tion Expression analysis DNaseI hypersensitivity 


  1. 1.
    Schouten, J.P., McElgunn, C.J., Waaijer, R., Zwijnenburg, D., Diepvens, F., and Pals, G. (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30, e57.PubMedCrossRefGoogle Scholar
  2. 2.
    Harteveld, C.L., Voskamp, A., Phylipsen, M., Akkermans, N., den Dunnen, J.T., White, S.J., and Giordano, P.C. (2005) Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alpha- and beta-thalassaemia characterised by high resolution multiplex ligation-dependent probe amplification. J Med Genet 42, 922–31.PubMedCrossRefGoogle Scholar
  3. 3.
    White, S.J., Vink, G.R., Kriek, M., Wuyts, W., Schouten, J., Bakker, B., Breuning, M.H., and den Dunnen, J.T. (2004) Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses. Hum Mutat 24, 86–92.PubMedCrossRefGoogle Scholar
  4. 4.
    Spek, C.A., Verbon, A., Aberson, H., Pribble, J.P., McElgunn, C.J., Turner, T., Axtelle, T., Schouten, J., Van Der Poll, T., and Reitsma, P.H. (2003) Treatment with an anti-CD14 monoclonal antibody delays and inhibits lipopolysaccharide-induced gene expression in humans in vivo. J Clin Immunol 23, 132–40.PubMedCrossRefGoogle Scholar
  5. 5.
    Eldering, E., Spek, C.A., Aberson, H.L., Grummels, A., Derks, I.A., de Vos, A.F., McElgunn, C.J., and Schouten, J.P. (2003) Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 31, e153.PubMedCrossRefGoogle Scholar
  6. 6.
    Nygren, A.O., Ameziane, N., Duarte, H.M., Vijzelaar, R.N., Waisfisz, Q., Hess, C.J., Schouten, J.P., and Errami, A. (2005) Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 33, e128.PubMedCrossRefGoogle Scholar
  7. 7.
    Leonhard, W.N., Roelfsema, J.H., Lantinga-van Leeuwen, I.S., Breuning, M.H., and Peters, D.J. (2008) Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice. BMC Biotechnol 8, 18.PubMedCrossRefGoogle Scholar
  8. 8.
    Ohnesorg, T., Eggers, S., Leonhard, W.N., Sinclair, A.H., and White, S.J. (2009) Rapid high-throughput analysis of DNaseI ­hypersensitive sites using a modified MLPA approach. BMC Genomics 10, 412.PubMedCrossRefGoogle Scholar
  9. 9.
    Lalic, T., Vossen, R.H., Coffa, J., Schouten, J.P., Guc-Scekic, M., Radivojevic, D., Djurisic, M., Breuning, M.H., White, S.J., and den Dunnen, J.T. (2005) Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 13, 1231–4.PubMedCrossRefGoogle Scholar
  10. 10.
    Zeng, F., Ren, Z.R., Huang, S.Z., Kalf, M., Mommersteeg, M., Smit, M., White, S., Jin, C. L., Xu, M., Zhou, D.W., Yan, J.B., Chen, M.J., van Beuningen, R., Huang, S.Z., den Dunnen, J., Zeng, Y.T., and Wu, Y. (2008) Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients. Hum Mutat 29, 190–7.PubMedCrossRefGoogle Scholar
  11. 11.
    Notini, A.J., Craig, J.M., and White, S.J. (2008) Copy number variation and ­mosaicism. Cytogenet Genome Res 123, 270–7.PubMedCrossRefGoogle Scholar
  12. 12.
    White, S.J., Vissers, L.E., Geurts van Kessel, A., de Menezes, R.X., Kalay, E., Lehesjoki, A.E., Giordano, P.C., van de Vosse, E., Breuning, M.H., Brunner, H.G., den Dunnen, J.T., and Veltman, J.A. (2007) Variation of CNV distribution in five different ethnic populations. Cytogenet Genome Res 118, 19–30.PubMedCrossRefGoogle Scholar
  13. 13.
    Groth, M., Szafranski, K., Taudien, S., Huse, K., Mueller, O., Rosenstiel, P., Nygren, A.O., Schreiber, S., Birkenmeier, G., and Platzer, M. (2008) High-resolution mapping of the 8p23.1 beta-defensin cluster reveals strictly concordant copy number variation of all genes. Hum Mutat 29, 1247–54.PubMedCrossRefGoogle Scholar
  14. 14.
    Aten, E., White, S.J., Kalf, M.E., Vossen, R.H., Thygesen, H.H., Ruivenkamp, C.A., Kriek, M., Breuning, M.H., and den Dunnen, J.T. (2008) Methods to detect CNVs in the human genome. Cytogenet Genome Res 123, 313–21.PubMedCrossRefGoogle Scholar
  15. 15.
    Bayley, J.P., Grimbergen, A.E., van Bunderen, P.A., van der Wielen, M., Kunst, H.P., Lenders, J.W., Jansen, J.C., Dullaart, R.P., Devilee, P., Corssmit, E.P., Vriends, A.H., Losekoot, M., and Weiss, M.M. (2009) The first Dutch SDHB founder deletion in ­paraganglioma-pheochromocytoma patients. BMC Med Genet 10, 34.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  • Thomas Ohnesorg
    • 1
  • Erin Turbitt
    • 1
  • Stefan J. White
    • 1
  1. 1.Molecular Development, Murdoch Children’s Research InstituteRoyal Children’s HospitalMelbourneAustralia

Personalised recommendations