Methylation-Specific MLPA® (MS-MLPA)
Methylation-specific MLPA (MS-MLPA) is a semi-quantitative method for methylation profiling. MS-MLPA is a variant of the MLPA technique in which copy number detection is combined with the use of a methylation-sensitive restriction enzyme (1). MS-MLPA is now widely used for the detection of epigenetic alterations. One of its major applications is methylation detection in imprinting diseases like Prader Willi/Angelman and Beckwith Wiedemann/RSS syndrome (2-4). MS-MLPA is also frequently used in tumour analysis (5-6), where it is used to examine the transcriptional inactivation of tumour suppressor genes which may lead to tumour progression (5) or resistance to chemotherapeutic agents (6). Detection of aberrant methylation patterns can thus be used to examine the type of tumour more closely.

The MS-MLPA protocol is very similar to the standard MLPA method, except that each MS-MLPA reaction generates two samples: one undigested sample for copy number detection and one digested sample for methylation detection. The MS-MLPA procedure can be divided in five steps (Figure 1): 1. DNA denaturation and hybridisation of MLPA probes; 2. ligation and digestion; 3. PCR; 4. separation of amplification products by capillary electrophoresis and 5. data analysis. MS-MLPA probes for methylation detection resemble other MLPA probes, except that their target sequence contains the restriction site of the methylation-sensitive endonuclease HhaI. After hybridisation, the reaction is split into two tubes: one tube is processed as a standard MLPA reaction, providing information on copy number changes (left panels in Figure 1). The other is incubated with the HhaI endonuclease meanwhile hybridized probes are ligated (right panels in Figure 1). Hybrids of probes and unmethylated sample DNA are digested by the HhaI enzyme. Digested probes cannot be amplified exponentially during the PCR and hence will not produce a signal during capillary electrophoresis. In contrast, if the sample DNA is methylated, the DNA-probe hybrids are protected against HhaI digestion and the ligated probes will generate a peak.


Figure 1: MS-MLPA reaction.

References
  1. Homig-Holzel C. and Savola, S. (2012). Multiplex Ligation-dependent Probe Amplification (MLPA) in Tumor Diagnostics and Prognostics.Diagn Mol Pathol.
  2. Nygren AO, et al. (2005) Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences Nucleic Acids Res 33, 14:e128
  3. Bittel, D.C., et al. (2007). Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities Genet Test 11, 467-475.
  4. Dikow, N., et al. (2007). Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA Mol Cell Probes 3, 208-215.
  5. Procter, M., et al. (2006). Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification Clin Chem 52, 1276-83.
  6. Hess, C.J., et al. (2008) Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia Leuk.Lymphoma 49, 1132-1141.
  7. Jeuken, J., et al. (2007). MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas. Lab Invest. 87(10):1055-65.
New Products
P484-ALPL
Hypophosphatasia (HPP)
Improved Products
P278-PCCA-PCCB
Propionic acidemia
P158-JPS
Juvenile polyposis syndrome (JPS)
P072-MSH6-MUTYH
Lynch syndrome; MUTYH-associated polyposis (MAP)
P225-PTEN
PTEN hamartoma tumor syndrome, Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, PTEN-related proteus syndrome, Proteus-like syndrome; (Tumour analysis for research use only)
P128-CYP450
Cytochrome P450
P021-SMA
Spinal muscular atrophy (SMA)
P409-RASA1-EPHB4
capillary malformations, Parkes Weber syndrome
P419-CDKN2A/2B-CDK4
Familial melanoma
P389-MLL2
Kabuki syndrome
P324-22q11
22q11
P202-IKZF1-ERG
Acute lymphoplastic leukemia (ALL), CVID, CML
Newsletter  |  Home  |  Privacy Policy  |  Terms and Conditions  |  Search  |  Sitemap  |  Copyright © 2019 MRC-Holland