MLPA® - an introduction
MLPA (Multiplex Ligation-dependent Probe Amplification) is a multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide (1). The MLPA technique is easy to use and can be performed in many laboratories, as it only requires a thermocycler and capillary electrophoresis equipment. Up to 96 samples can be handled simultaneously, with results being available within 24 hours. Although for most hereditary conditions, (partial) gene deletions or duplications account for less than 10 % of all disease-causing mutations, for many other disorders this is 10 to 30 % (2-8) or even higher still (9, 10). The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.
Advantages of MLPA
Using MLPA for copy number detection offers many advantages over other techniques. First of all, methods which were primarily developed for detecting point mutations, such as sequencing and DHPLC, generally fail to detect copy numbers changes. Southern blot analysis, on the other hand, will reveal many aberrations but will not always detect small deletions and is not ideal as a routine technique. Although well-characterised deletions and amplifications can be detected by PCR, the exact breakpoint site of most deletions is unknown. Furthermore, when comparing MLPA to FISH, MLPA not only has the advantage of being a multiplex technique, but also one in which very small (50-70 nt) sequences are targeted, enabling MLPA to identify the frequent, single gene aberrations which are too small to be detected by FISH. Moreover, MLPA can be used on purified DNA. Finally, as compared to array CGH, MLPA is a low cost and technically uncomplicated method. Although MLPA is not suitable for genome-wide research screening, it is a good alternative to array-based techniques for many routine applications. The over 300 probe sets now commercially available are dedicated to applications ranging from the relatively common (Duchenne, DiGeorge syndrome, SMA) to the very rare (hereditary pancreatitis, Antithrombin deficiency, Birt-Hogg-Dube syndrome).
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.
The MLPA reaction can be divided in five major steps: 1) DNA denaturation and hybridisation of MLPA probes; 2) ligation reaction; 3) PCR reaction; 4) separation of amplification products by electrophoresis; and 5) data analysis (Figure 1). 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). Only when the two probe oligonucleotides are both hybridised to their adjacent targets can they be ligated during the ligation reaction (Figure 1 - step 2). Because only ligated probes will be exponentially amplified during the subsequent PCR reaction (Figure 1 - step 3), 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 4). 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.
A few variations on MLPA have been developed. RT-MLPA (Reverse Transcriptase MLPA) can be used for mRNA profiling (11). As the ligase enzyme cannot ligate probes which are bound to RNA, the RT-MLPA procedure starts with the reverse transcription of mRNA into cDNA. After this, RT-MLPA continuous as a normal MLPA reaction. Another variation is Methylation-Specific MLPA (MS-MLPA), which can be used for both copy number quantification and methylation profiling (12). MS-MLPA has proven to be a very useful method for the detection of imprinting diseases (13-15) and for the analysis of methylation aberrations in tumour samples (16, 17).
SALSA MLPA kits for a rapidly growing number of applications are available from MRC-Holland (Amsterdam, the Netherlands). A list of available SALSA MLPA kits can be found here.
- Schouten JP et al. (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification Nucleic Acids Res 30, e57.
- Aretz, S. et al. (2007). High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome J Med Genet. 44, 702-709.
- Redeker, E.J., et al. (2008). Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders Mol Vis 14, 836-840.
- Kanno, J., et al. (2007). Genomic deletion within GLDC is a major cause of non-ketotic hyperglycinaemia J Med Genet 44, 3.
- Aldred, M.A., et al. (2006). BMPR2 gene rearrangements account for a significant proportion of mutations in familial and idiopathic pulmonary arterial hypertension Hum Mutat 2, 212-213.
- Kluwe, L., et al. (2005). Screening for large mutations of the NF2 gene Genes Chromosomes Cancer 42, 384-391.
- Michils, G., et al. (2005). Large deletions of the APC gene in 15% of mutation-negative patients with classical polyposis (FAP): a Belgian study Hum Mutat 2, 125-34.
- Taylor, C.F., et al. (2003). Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA Hum Mutat 6, 428-33.
- Depienne, C., et al. (2007). Exon deletions of SPG4 are a frequent cause of hereditary spastic paraplegia J Med Genet 44, 281-284.
- Beetz, C., et al. (2006). High frequency of partial SPAST deletions in autosomal dominant hereditary spastic paraplegia Neurology 67, 1926-1930.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.