General Questions on MLPA
1. What is MLPA?
Multiplex Ligation-dependent Probe Amplification (MLPA®) is a method which detects aberrant copy numbers of up to 45 specific nucleic acid sequences in one simple PCR reaction, using a single PCR primer pair. MLPA reactions require only 20 ng of human chromosomal DNA or 10-120 ng total RNA. Applications include the detection of exon deletions/duplications in e.g. the human BRCA1, MSH2 and MLH1 genes; detection of trisomies such as Down syndrome; characterisation of chromosomal aberrations in cell lines and tumour samples; SNP and mutation detection; DNA methylation analysis and relative quantification of mRNA.
A conventional multiplex PCR requires one pair of primers for each fragment to be amplified. These primers are present in large amounts during the reaction, resulting in various problems. Firstly, since the efficiency of different primer pairs usually varies, it is difficult to use conventional multiplex PCR for relative quantification of target sequences. Furthermore, small differences in reaction conditions often result in large differences in the results obtained.
The reason that MLPA reactions are more robust is that all fragments are amplified with the use of a single PCR primer pair. How is this possible? The trick of MLPA is that it is not the sample DNA that is amplified, but the MLPA probes that are added to the sample. Each MLPA probe consists of two paired/matching oligonucleotides, each one containing one of the PCR primer sequences plus a sequence complementary to the DNA target sequence. These two probe oligonucleotides hybridise to immediately adjacent target sites. It is only when the two probe oligonucleotides are both hybridised to their target that they can be ligated by into a single probe, containing both the forward and reverse primer sequence. While these ligated probes are amplified exponentially during PCR reaction, the individual non-ligated probe oligonucleotides are not. The number of probe ligation products of one probe therefore depends on the number of target sequences in the sample.
Following the PCR, the resulting amplification products are separated by sequencing electrophoresis. An MLPA probe set is designed so that the length of each of its amplification products is unique. The length increases in a stepwise-fashion by 6 or 9 nucleotides, with the total size range lying between 120-480 nucleotides. This size range provides an optimal fragment separation and a low background on sequence type gels.
Besides a (capillary) electrophoresis equipment, only a thermocycler is required to perform MLPA. MLPA reactions can be performed as a one tube reaction, as non-ligated oligonucleotides do not need to be removed.
2. What are the advantages of MLPA?
- The MLPA technique can be used for many different applications. The only difference between different MLPA assays is the probemix; all MLPA reagents are the same.
- MLPA is multiplex: one reaction provides information on up to 50 targets. For most applications, this is sufficient to answer the specific questions asked by a physician.
- MLPA reactions are relatively cheap: the price for an MLPA probemix + reagent kit (100 reactions) is € 1242. This includes all reagents (probemix, buffers, ligase, polymerase, dNTPs and labelled PCR primers).
- MLPA is reproducible and easy to perform, and large numbers of samples can be tested simultaneously.
- MLPA is sensitive: only 20 ng of human DNA is required, and results do not depend on the amount of sample DNA used.
- MLPA can distinguish sequences differing in only a single nucleotide and is able detect small copy number differences - e.g. 3 vs. 2 copies of a given gene sequence in a DNA sample.
- MLPA detects a sequence of only ~60 nucleotides and can thus be used to detect deletions or duplications of a single exon.
- Investment costs are low for most users: the necessary equipment for MLPA consists of a thermocycler and capillary sequence equipment, both of which are present in most molecular biology laboratories. Although capillary electrophoresis is preferred, MLPA has also been used in combination with LICOR and ABI 373 and 377 slab gels.
3. What are the limitations of MLPA?
- MLPA reactions are more sensitive to contaminants (PCR inhibitors such as small remnants of phenol) than conventional PCR reactions. However, it will be quickly detected whether this has been the case, as it is predominantly those amplification products with lower average peak areas (usually the longer probes) whose signals will be reduced. Moreover, good results can be obtained on DNA extracted from formaldehyde-treated paraffin-embedded tissues.
- Developing the type of MLPA probe sets sold by MRC-Holland is complicated, expensive and time-consuming. Each probe requires the design and preparation of a phage M13 clone, purification of its single-stranded DNA and subsequent digestion with expensive restriction endonucleases. Nevertheless, hundreds of different SALSA MLPA probemixes are already available from MRC-Holland and it is always possible to send in requests for new probe mixes. For research purposes, it is possible to design a small number of completely synthetic probes, resulting in amplification products with a length between 100 and 140 nt. Guidelines and sequences of the PCR primers are available here.
- In contrast to FISH, MLPA cannot yet be used to investigate single cells. MLPA analysis of DNA samples from cell mixtures will give the average copy number per cell. In case of tumour analysis, it will be difficult to detect deletions of a certain gene if the sample from which the DNA was derived contained less than 50% cancer cells.
- MLPA is primarily a method which identifies genomic deletions/duplications; it is not a suitable method to detect unknown point mutations. Nevertheless, MLPA is able to discriminate known (point) mutations, as probes can be designed so that the ligation site is located directly at the site of the (point) mutation. Ligation will then only occur on non-mutated sequences, resulting in a decreased fluorescent signal in the case of mutated DNA.
- MLPA probes are not only sensitive to complete deletions of a probe’s target sequence, but also to small deletions, insertions and mismatches. Any mismatch in the probe’s target site can theoretically affect the probe signal. (How strong the destabilising effect is, depends on the location of the mismatch, the type of mismatch and the Tm of the probe’s hybridising sequence, among others). This means that a (non-pathogenic) polymorphism can also result in a decreased probe signal, thus mimicking a deletion. When designing MLPA probes, MRC-Holland excludes any sequences in which SNPs or other polymorphisms are found using the latest genetic search engines. However, as new polymorphisms are continuously being discovered -sometimes very localised ones- it is impossible to exclude this in all cases. This is why it is recommend to confirm all MLPA findings with another method, especially in the case of single probe deletions.
4. Which equipment is recommended for MLPA analysis?
At our labs we use Thermo Scientific PCR tubes with the following model numbers:
For the PCR reaction it is important to use a thermocycler which offers the possibility of adding reagents while samples are in the apparatus. The Stratagene Robocycler is therefore more difficult to use. Following the PCR reaction, samples are analysed by capillary electrophoresis. When using slab gel electrophoresis, one should preferably use an apparatus that provides a scan of the total gel, such as the LICOR. Different sequencers require different fluorescent labels. The SALSA MLPA PCR primers offered by MRC-Holland are available with various fluorescent labels. FAM-labelled primers are recommended for all Applied Biosystems sequencers, whereas the Cy5 label is used for Beckman sequencers. Also available are unlabeled PCR primers that can be isotopically labelled by kinase treatment.
5. Can I use the Qiaxcel system for MLPA?
We do not recommend using a Qiaxcel system for MLPA fragment analysis. The apparatus has insufficient resolving power for a typical MLPA probe set, not enough to resolve 50 different fragments with a length 100 nt -500 nt. Secondly, MRC-Holland does not test SALSA MLPA products with double-strand amplification products, as the PCR in the standard MLPA protocol ends with denaturation step and thus single-strand amplicons. Hence, we cannot guarantee that the double-strand DNA fragments yielded by Qiaxcel, or any other ethidium-bromide-based system, will be separated properly. Because of these two aspects, we recommend using a capillary sequencer instead.
6. Can I use MLPA on an ALF express?
It is not recommended. It is possible to detect 40 different fragments with an ALF express. However, the problem with this apparatus is that the detectors are fixed. Hence, gels that do not run perfect will give problems, as e.g. sample 37 might end up in front of detector 38. Moreover, problems with quantification may occur as smaller fragments may be detected perfectly, while longer fragments may migrate predominantly between the two different detectors. In case you have access to a capillary sequence, this would be strongly preferred. In case the ALF express is used, however, we recommend quantifying peaks by comparing them to nearby peaks of similar size.
8. Is it possible to use MLPA for testing the methylation status of a specific site?
Yes. We have several methylation-specific MLPA probemixes available including the ME001 for tumour suppressor genes and ME028 for Prader-Willi syndrome (PWS)/Angelman syndrome (AS). MLPA probes for methylation quantification are similar to normal MLPA probes. The main difference is that the sequence detected by the MS-MLPA probes contains a recognition site for the methylation-sensitive restriction enzyme HhaI. During the MS-MLPA procedure, the complex which is formed after hybridisation of the probes to the sample DNA will be digested by the HhaI restriction endonuclease. Probes which are hybridised to an unmethylated site are digested and will therefore not produce a signal. In contrast, if the DNA target sequence detected by the probe is methylated, the sequence is protected against digestion and the probe will generate a signal. More information about the MS-MLPA technology can be found here, while an overview of MS-MLPA probemixes is available here. Please note that the HhaI enzyme, which is required for MS-MLPA reactions, is not included in the MLPA probemixes!
9. Is it possible to detect point mutations with MLPA?
MLPA is primarily a method which identifies deletions/duplications - it is not a suitable method to detect unknown point mutations. Nevertheless, MLPA is able to discriminate known point mutations, as probes can be designed so that the ligation site of a probe is located directly at the site of the point mutation. Ligation will then only occur on if the target sequence is the perfect reverse complement of the probe sequence, resulting in a decreased fluorescent signal in the case of a mismatched DNA sequence. Probes can be designed either to detect the wild type sequence or the SNP/point mutation in question.
10. What is the difference between DNA, RNA, and Methylation MLPA probes?
DNA and Methylation probes are used for the detection and quantification of chromosomal DNA sequences. The two probe oligonucleotides hybridise to immediately adjacent target DNA sequences. Methylation probes are similar to DNA probes, except that they contain a recognition site for the methylation-sensitive restriction endonuclease HhaI and are usually located within CpG islands. RNA probes are specific for cDNA sequences derived from mRNAs, and most probes will not generate any signal on contaminating DNA. This is because the hybridising parts of the two oligonucleotides of an RNA probe are located in adjacent exons, which are separated by an intron in chromosomal DNA.
11. Is it possible to use SALSA MLPA probes on DNA from other organisms?
No. The chance of mismatches close to the ligation site is very high. Mismatches close to the ligation site will prevent ligation and thus the formation of amplifiable probes. Reducing the hybridisation temperature will not make any difference.
12. Can I use SALSA MLPA kits in diagnostics?
MRC-Holland emphasises that MLPA products are intended for research purposes only, since the SALSA MLPA products are not FDA/CE certified. Nonetheless, some laboratories use MLPA products for diagnostic purposes under their own responsibility. If you choose to use our SALSA MLPA products in a diagnostic setting, for instance because no other suitable method is available, MLPA products should be validated in house using your own quality system. MRC-Holland specifies that these kits are for research purposes only, and users take full responsibility when using MLPA test results for clinical decisions.
13. Do I need to confirm MLPA results with other methods?
Single MLPA probe results should always be confirmed by another method, as mutations and/or polymorphisms under the probe hybridising sequence may also result in a reduced relative probe signal. For some of our SALSA MLPA kits, confirmation kits are available. If possible, confirm a deletion with other MLPA probes, Southern blots, long range PCR and/or FISH. Duplication are generally harder to verify but can sometimes be confirmed by Southern blots. False duplications can be the result of experimental errors (for instance when a reference sample is used for normalisation which has a reduced signal for the probe in question), contamination of the sample or the possibility that the probe recognizes an unknown pseudogene. This risk should be reduced during (synthetic) probe design. MRC-Holland always screens new probes for any homology with known pseudogenes. It is furthermore recommended to use multiple reference samples positioned throughout your assay to measure the probe variability in each experiment.
14. Can I have my samples tested at MRC-Holland?
No. MRC-Holland does not have a testing facility.
15. Can I order synthetic probes from MRC-Holland?
No. We don’t design or produce synthetic probes for customers. Synthetic probes consist of two synthetic oligonucleotides that can be ordered from your own oligo supplier. We recommend using our SALSA MLPA reference probemixes P200 or P300. These probemixes contain control fragments and reference probes that can be used in combination with your own synthetic probes. Alternatively, only an MLPA reagent kit (EK1 kit) can be ordered from MRC-Holland; these kits are recommended only for users who are already experienced with MLPA probe design. Please note that it is not possible to order MLPA reagent kits in quantities smaller than 100 reactions. More information on designing synthetic probes can be found here.
16. What are the different version numbers?
To make sure changes in a probemix are noticed by its users, MRC-Holland uses version numbers. A new probemix always has version number A1. Letters denote major versions, whereas numbers are used for subversions. To denote minor changes such as the addition of the D-control fragments to the probemix, the number after the letter changes, for instance from A1 to A2. To specify major changes in the probemix, like the addition of a new probe, we change the letter, for example from A3 to B1. One version can have various lots if in the meantime nothing has changed content of the probemix. All changes made to a probemix are indicated in the probemix description.
17. Is MLPA a patented technology?
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18. Why does MRC-Holland occasionally change the exon numbering or gene name used in its product materials?
One gene may be known under various aliases. MRC-Holland has chosen to use only those gene names that have been officially approved by the HUGO Gene Nomenclature Committee. Any alternative names can be easily found by doing a search on www.genenames.org.
A similar policy is applied to exon numbering. Although for most genes, exon numbering is relatively simple, there are also genes for which various numbering systems exist. Usually, these are genes for which as older (but factually incorrect) exon numbering had already become in widespread use, or genes which consists both of coding and noncoding exons or have rare transcript variants. MRC-Holland has chosen to use only the exon numbering system of National Center for Biotechnology Information (NCBI) in all its product materials. When NCBI changes its exon numbering, any product materials will also be changed at the moment that the product in question is updated.
The changing of gene names and exon numbering in an inevitable consequence of the increased knowledge of genes and their functions. By adhering to a single, widely recognised system for both gene nomenclature and exon numbering, MRC-Holland aims to have transparency and clarity in its product materials. Nevertheless, MRC-Holland realises that changing gene names and exon numbers may cause confusion. MRC_Holland will therefore always include a note if exon numbering or a gene name has been altered to alert customers of this change