How Does MLPA® Work?
MLPA reaction
Typical for MLPA is that it is not target sequences that are amplified, but MLPA probes that hybridise to the target sequence. In contrast to a standard multiplex PCR, a single pair PCR primers is used for MLPA amplification. The resulting amplification products of a SALSA MLPA kits range between 130 and 480 nt in length and can be analysed by capillary electrophoresis. Comparing the peak pattern obtained to that of reference samples indicates which sequences show aberrant copy numbers. Read more about the advantages of MLPA on this page.

The MLPA reaction can be divided in five major steps: 1&2) DNA denaturation and hybridisation of MLPA probes; 3) ligation reaction; 4) PCR reaction; 5) separation of amplification products by electrophoresis; and 6) data analysis (Figure 1; or see the video at the bottom of this page). During the first step, the DNA is denatured and incubated overnight with a mixture of MLPA probes. MLPA probes consist of two separate oligonucleotides, each containing one of the PCR primer sequences. The two probe oligonucleotides hybridise to immediately adjacent target sequences (Figure 1 - step 1/2). Only when the two probe oligonucleotides are both hybridised to their adjacent targets can they be ligated during the ligation reaction (Figure 1 - step 3). Because only ligated probes will be exponentially amplified during the subsequent PCR reaction (Figure 1 - step 4), the number of probe ligation products is a measure for the number of target sequences in the sample. The amplification products are separated using capillary electrophoresis (Figure 1 - step 5). Probe oligonucleotides that are not ligated only contain one primer sequence. As a consequence, they cannot be amplified exponentially and will not generate a signal. The removal of unbound probes is therefore unnecessary in MLPA and makes the MLPA method easy to perform.


Figure 1: MLPA reaction.

Video

Alternative ways to watch the video can be found in this knowledgebase article (opens in a new window) if the embedded video above does not work. The knowledgebase also contains other educational material.

MLPA variations
A few variations on MLPA have been developed. Methylation-Specific MLPA (MS-MLPA) can be used for both copy number quantification and methylation profiling (1). MS-MLPA has proven to be a very useful method for the detection of imprinting diseases (2-4) and for the analysis of methylation aberrations in tumour samples (5-6). RT-MLPA (Reverse Transcriptase MLPA) was developed for mRNA profiling (7), but MRC-Holland no longer sells products that utilize this technique.

References
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. Jeuken, J., et al. (2006). Multiplex ligation-dependent probe amplification: a diagnostic tool for simultaneous identification of different genetic markers in glial tumors J Mol Diagn 4, 433-443.
  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. Eldering, E., et al. (2003). Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways Nucleic Acids Res 31, e153.
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