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General MLPA FAQ
What is MLPA ?
What are the advantages of MLPA?
What are the disadvantages of MLPA?
Which equipment is recommended for MLPA analysis?
Can I use a thermal cycler without heated lid?
Can I use MLPA on an ALF express?
Are there MLPA kits available for the detection on agarose gels?
Is it possible to use MLPA to test the methylation status of a specific site?
Is it possible to detect point mutations with MLPA?
What is the difference between SALSA DNA, RNA, & Methylation probes?
Are SALSA MLPA kits compatible with whole genome amplified (WGA) DNA?
Is it possible to use SALSA probes on DNA from other organisms?
Can I have my samples tested at MRC-Holland?
Is MLPA a patented technology?
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1. What is MLPA ?
Basically, 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's syndrome; characterisation of chromosomal aberrations in cell lines and tumour samples; SNP and mutation detection; DNA methylation analysis and relative quantification of mRNAs.
An ordinary 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. Since the efficiency of different primer pairs usually varies, it is difficult to use ordinary multiplex PCR amplification 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. But how can one amplify and quantify 45 different sequences using just one pair of primers? With a traditional multiplex PCR, this would be impossible. The trick of MLPA is that no sample DNA is amplified, but only probes that are added to the sample. MLPA probes consists of two different oligonucleotides, each containing one of the PCR primer sequences as well as a sequence complementary to the target sequence. The two probe oligonucleotides hybridise to immediately adjacent target sequences. It is only when the two probe parts are both hybridised to their target sequence that they can be ligated to each other by a thermo stable ligase. Whereas these ligated probes will be amplified exponentially during PCR reaction, the individual non-ligated probe oligonucleotides will not. The number of probe ligation products of one probe depends on the number of target sequences in the sample.
The resulting PCR amplification products are subsequently separated by sequence type electrophoresis. MLPA tests are designed in a way that the length of each amplification product 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.
Apart from electrophoresis equipment, only a thermocycler is required to perform an MLPA reaction. MLPA reactions can be performed as a one tube reaction, as non-ligated probes do not need to be removed.
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2. What are the advantages of MLPA?
The advantages of MLPA are:
* The MLPA technique can be used for many different applications; the only difference between different assays is the probe mix used.
* MLPA is multiplex: one reaction provides information on up to 45 targets. For most applications, this is sufficient to answer the specific questions asked by a physician.
* MLPA reactions are relatively cheap: the current price for an MLPA kit for 100 reactions is €1060. This includes all reagents (probemix, buffers, ligase, polymerase, dNTP's 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 that differ 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 the complex mixture of the human genome.
* 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 DNA sequencing electrophoresis 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.
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3. What are the disadvantages of MLPA?
* MLPA reactions are more sensitive to contaminants (PCR inhibitors such as small remnants of phenol) than ordinary 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 (e.g. the longer probes) that will show reduced signals. Moreover, please note that good results are obtained on DNA extracted from formaldehyde-treated paraffin embedded tissues (see the support section on www.mlpa.com).
* Developing MLPA probe mixes as sold by MRC-Holland is complicated, expensive and time-consuming. Each probe requires the design and preparation of a phage M13 clone, the purification of its single stranded DNA, and digestion with expensive restriction endonuclease. Nevertheless, more than 120 different MLPA kits 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 130 nt. Guidelines and sequences of the PCR primers are available on www.mlpa.com.
* 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 tumor 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/insertions - 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.
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4. Which equipment is recommended for MLPA analysis?
We use Biometra Uno II thermocyclers (96 x 0.2 ml) for the MLPA reactions. It is important that some additions to the reactions can be made while the samples are in the thermocycler. The Stratagene Robocycler is therefore more difficult to use. Separation and relative quantification of probe amplification products is performed at MRC-Holland with the use of Beckman CEQ8000 capillary sequencers (Cy5.0 labelled PCR primers). Most clients use Applied Biosystems capillary sequencers with FAM labelled PCR primers. When using slab gel electrophoresis, one should preferably use an apparatus that provides a scan of the total gel, such as the LICOR. The use of an ALF Express with fixed detectors is not recommended.
MLPA probemixes can not yet be used on Caliper or Agilent 2100 bioanalyzers. At this moment the microfluidics systems have insufficient separating power to distinguish 45 peaks in the 130-500 nt range. In addition, the SALSA MLPA probemixes from MRC-Holland have been optimised for good separation of all fragments in denaturing conditions whereas the microfluidics systems use detection of double stranded nucleic acids.
Different thermocyclers require different fluorescent labels. The SALSA PCR primers offered by MRC-Holland are available with various fluorescent labels. The FAM labelled primer is 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.
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5. Can I use a thermal cycler without heated lid?
No. But a cheap clothes iron can replace the heated lid!
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6. Can I use MLPA on an ALF express?
Yes, 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 the smaller fragments might be detected perfectly, while the longer fragments might 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 to quantify peaks by comparing them to nearby peaks of similar size.
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7. Are there MLPA kits available for the detection on agarose gels?
Yes. We have a small number of MLPA kits available for the detection on agarose gels (P012 Her2-neu ; P071-P078 DMD). These probesets contain only 10-14 probes and are developed especially for labs in countries which cannot afford an electrophoresis type sequencer. For more information please e-mail us: info@mlpa.com.
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8. Is it possible to use MLPA for testing the methylation status of a specific site?
Yes. We have several methylation-specific MLPA kits available, such as ME001 for tumour suppressor genes and ME028 for Prader-Willi syndrome (PWS) and 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 sequence for the methylation-sensitive restriction enzyme HhaI. During the MS-MLPA procedure, the complex which is formed after hybridising of the probes to the sample DNA will be digested by the HhaI restriction endonuclease. Probes which are hybridized to an unmethylated site are digested and will therefore not produce a signal. In contrast, if the sample DNA sequence detected by the probe IS methylated, that probe will generate a signal. More information about MS-MLPA can be found on our website ' support. Please note that the HhaI enzyme, which is required for MS-MLPA reactions, is not included in the MLPA kits!
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9. Is it possible to detect point mutations with MLPA?
MLPA is primarily a method which identifies genomic deletions/insertions - 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 non-mutated sequences, resulting in a decreased fluorescent signal in the case of mutated DNA. Alternatively probes can be made that will only generate a signal if one or both alleles contain the specific mutation.
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10. What is the difference between SALSA DNA, RNA, and Methylation probes?
DNA and Methylation probes are used for the detection and quantification of chromosomal DNA sequences. The hybridizing sequences of the two probe oligonucleotides are immediately adjacent on the target DNA sequence. Methylation probes are similar to DNA probes, except that they contain a recognitionsitefor a methylation sensitive restriction endonuclease such as HhaI and HpaII and are usually located within CpG islands. Methylation probes are usually intended for methylation quantification of CpG sequences in these 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 of course separated by an intron in chromosomal DNA.
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11. Are SALSA MLPA kits compatible with whole genome amplified (WGA) DNA?
No, the reproducibility of whole genome amplified (WGA) DNA for small fragments (60 nt) as used in MLPA is insufficient.
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13. Can I have my samples tested at MRC-Holland?
No. MRC-Holland does not have a testing facility. MRC-Holland sells kits to labs, which can use MLPA for their research or to aid diagnostics.
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14. Is MLPA a patented technology?
MLPA products are the subject of U.S. patent no. 6955901 and other pending patent applications owned by DLH Octrooien B.V.
The purchase price of the MRC-Holland MLPA kits includes limited, non-transferable, rights for institutions to use only the purchased amount of the product for up to but no more than 5000 MLPA reactions per year per site to practice Multiplex Ligation-dependent Probe Amplification solely for internal research purposes. In particular, any distribution of SALSA vectors or vectors derived from SALSA vectors to third parties is not permitted without written consent from DLH Octrooien B.V.
Detection and quantification of certain nucleic acid sequences may require other licenses in certain countries. Purchase of SALSA reagents, probes, vectors or kits does not imply any other license for patents or patent applications other than the MLPA patent application of DLH Octrooien B.V.
MRC-Holland reserves all other rights and in particular, the purchaser of this product cannot transfer or otherwise sell this product or its components to a third party, and no rights are conveyed to the purchaser to use the product or its components or products derived from its components for commercial purposes as defined below. For-profit institutions must obtain a license from MRC-Holland to acquire rights to use this product for any purpose other than those permitted above.
Commercial purposes means any activity for which a party receives consideration and may include, but is not limited to, (1) use of the product or its components or products derived from one or more of its components in manufacturing, (2) use of the product or its components or products derived from one or more of its components to provide a service, information or data, (3) use of the product or its components or products derived from one or more of its components for diagnostic purposes, (4) transfer or sell vectors or clones made with one or more of the SALSA probes or vectors, or (5) resell the product or its components or products derived from one or more of its components, whether or not such product or its components are resold for use in research.
"MLPA " and "SALSA " are trademarks from DLH Octrooien B.V. and should not be used in technical and advertising material such as brochures, catalogues and the like without written permission from DLH Octrooien B.V. More information about legal issues is available from this website.
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Copyright © 2005 - MRC-Holland
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12. Is it possible to use SALSA MLPA DNA 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 probe-molecules. Lowering hybridisation temperature will not make any difference.
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