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P044-NF2
Neurofibromatosis type 2 (NF2)
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P199-HEXA
Tay-sachs disease
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P214-COL2A1
Skeletal disorders, Achondrogenesis, Chondrodysplasia, Early onset familial Osteoarthritis, SED congenital, Langer-Saldino achondrogenesis, Kniest dysplasia, Stickler syndrome, Spondyloepimetaphyseal
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P280-SLC26A4
Pendred syndrome
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P026-Sotos
Sotos syndrome
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ME011-MMR
Mismatch repair genes (MMR)
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Sample treatment
Important notice regarding the use of Qiagen Biorobot (EZ1/Geno-M6/M48/M96).
Various MLPA users have reported problems when using DNA extracted by the Qiagen EZ1 or M48 robot. If you are using a Qiagen Biorobot, please read our recommendations here.
In this document, you can find information on the following:
- Sample quality - which sample factors can influence your MLPA?
- Dissolving samples - what is the best way to dissolve samples
- DNA/RNA extraction - advise on extraction methods
- Extra purification of contaminated DNA samples - getting rid of contaminants
In brief
Although the MLPA reaction is robust and reproducible, the treatment of DNA (storage, extraction method) can influence the MLPA peak pattern. Contaminants which remain after extracting or dissolving the DNA can affect DNA denaturation and/or amplification of MLPA probes.
Within a single experiment, one should only compare samples that were extracted by the same method, preferably by the same laboratory and of similar concentrations. As the effect of contaminants is generally consistent and reproducible, a bias caused by impurities will often be corrected for by the samples having undergone identical treatments.
However, when contaminants are present in large quantities or in different concentrations (something which may be the case even despite identical treatment and which is hard to check for), problems may arise. If you are working with older samples, samples that have been extracted using different methods or other samples which may contaminants, try adding less sample or do an extra purification step.
1. Sample quality
- To minimise the effects of contaminants, always use sample and reference DNA extracted with the same method and derived from the same source. For instance, when testing DNA extracted from FFPE breast tumour tissue, compare this to similarly extracted FFPE samples from healthy breast tissue (does not have to be from the same individual).
- MLPA allows a DNA concentration range of 20-500 ng in 5 ul; within this range, results on pure DNA samples are independent of the exact concentration. However, it is recommended to use similar DNA concentrations to minimize variation caused by possible contaminants.
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Long term (years) storage and repeated freeze-thaw cycles may influence sample quality and MLPA results.
- MLPA users reported that reference samples used for a prolonged time started showing aberrant results. Including such samples as a reference can skew up data normalisation. Aliquot regularly-used reference DNA samples and store these at -20 oC. After thawing, the vial in use can be stored at 4 oC for at least 6 months.
- Since MLPA probes recognise targets of only 50-80 nt long, MLPA is hardly influenced by DNA fragmentation. DNA extracted from formalin-fixed, paraffin-embedded (FFPE) samples can thus be used. Please see the advice below on how to extract DNA from these tissues.
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Contaminants:
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Contaminants that do not influence MLPA
- Some contaminants in DNA samples, such as proteins, do not influence MLPA. Therefore, MLPA can be used directly on lysates obtained by ProteinaseK digestion of cells or on DNA from FFPE samples.
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Contaminants causing incomplete denaturation
- Salt concentration in DNA samples should be low. At MRC-Holland, we dissolve and dilute DNA samples in TE. For long term storage, DNA is more stable in TE than in water. Salt concentrations (NaCl, KCl) higher than 60 mM will cause problems with the DNA denaturation. Dissolving in 1 x PCR buffer is strongly discouraged.
- The presence of other ions, such as Fe and Mg, can already cause denaturation problems at concentrations below 1 mM. A concentration of as little as 0.1 mM MgCl2 in the sample can even increase the denaturation temperature (Tm) by 12 oC (Eichhorn, 1962). For this reason, DNA should not be dissolved in 1 x PCR buffer.
- Incomplete DNA denaturation results in low signals of probes located close to CpG islands (see here for more information) and can be easily detected by low signals of the 88 and 96 nt D-control fragments.
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Contaminants inhibiting the PCR:
The MLPA PCR reaction is more sensitive to certain impurities than conventional PCR. The following can influence the PCR reaction:
- Ionic impurities such as iron (from blood cells or leaking from magnetic beads); ethanol, phenol and Trizol remnants all reduce polymerase activity. Not all probes react the same way to this reduced activity though: while most probes will not be affected, some may show a reduction in peak height whereas yet others will increase in signal. Laboratories which experienced problems with DNA samples reported that these could be contributed to left-over phenol and ethanol remnants.
- EDTA: DNA samples should also not contain more than 1 mM EDTA as EDTA binds Magnesium. A high concentration of Magnesium (present in Ligase buffer B and PCR buffer) is required for the ligation and PCR reaction.
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In case of sample DNA contamination:
- Use less sample DNA (20-50 ng): when the DNA concentrations are quite high you may choose to strongly dilute your samples, thereby also diluting the concentration of contaminants. Make sure you use at least 20 ng DNA.
- Do an extra purification step: clean contaminated samples by ethanol precipitation or silica columns. This is especially a good idea for old samples if enough DNA is available. More information on purification can be found here.
- Prolong the 98 oC denaturation step from 5 to 40 minutes.
- Finally, when the MLPA is properly performed and analysed, the probe standard deviation between samples should be a maximum of 10 % for the great majority of probes. When a larger variation is found, this may indicate differences between samples exist. When working with samples that (1) were extracted using different methods, (2) are old, partly evaporated and/or viscous or (3) which may contain contaminants, an extra purification step, such as a simple ethanol precipitation, may help. For DNA samples of lesser quality, using smaller volumes of sample DNA (around 50 ng) is often also beneficial.
2. Dissolving samples
The buffer used to dissolve DNA is important. The pH of the DNA preparation should be around 8.0 or 8.5 in order to prevent depurination during the initial heat treatment at 98°C.
We recommend the use of TE (10 mM Tris-HCl pH 8.2 + 0.1 mM EDTA) for dissolving and diluting DNA. Do not use more than 1 mM EDTA in the sample solution as it will bind Magnesium and may influence the ligation or PCR reaction! Tris buffers are much more acidic at elevated temperatures and are thus not recommended.
The use of 1 x PCR buffer to dissolve DNA is strongly discouraged! This buffer contains both Magnesium and salt, the combination of which can prevent the denaturation of CG-rich areas. Our test results have shown that when the DNA is dissolved in 1 x PCR buffer (containing 50 M KCl, 1.5 mM MgCl2), most CpG islands are no longer completely denatured at a heat treatment of 98 °C. Although in every sample, there will be some sequences in CpG islands that are still amplified (as there will always be some strands that are more fragmented and which can thus be denatured), the quantity measured will strongly depend on the degree of fragmentation of the DNA. Hence, when comparing strongly fragmented (degraded) DNA with less fragmented DNA, the former will appear to have a much higher copy number of sequences located close to CpG islands. The quantification of sequences located less than 5-10 Kb from CpG islands is thus severely compromised by the use of this buffer.
3. DNA/RNA extraction
MLPA does not require a special for DNA extraction method or kit. Many users obtain good results with simply salting out or by phenol/chloroform extraction. Various Qiagen kits are also widely used with good results..
Problems with automated DNA extraction methods, such as Roche Magnapure and Qiagen’s EZ1 robot have been reported. It seems the first has been improved, as we now receive fewer comments about it. The Qiagen EZ1 BioRobot leaves too much salt in the samples (reports from the UK, Ireland and own testing), causing MLPA probes that are salt-sensitive to become variable. Please read our recommendations here. DNA extracted from heparin-treated blood can also give more problems as it is difficult to remove from DNA preparations. Below you can find information and protocols for some suitable extraction methods.
Please note that at MRC-Holland, we do not extract DNA. The information in this protocol is courtesy of MLPA users. Please e-mail us at info@mlpa.com about your experiences so we can continue to improve these guidelines.
DNA extraction from blood
| Method |
Comments |
Advantages |
Disadvantages |
| Phenol/chloroform |
Most frequently used |
- Good DNA quality
- Inexpensive
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- Time-consuming
- Phenol may remain in sample
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| Salting out method |
Frequently used |
- Good results
- Inexpensive
- Rapid
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| Standard column isolation |
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| Qiagen (Gentra) kit |
Frequently used |
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| Qiagen (Mini blood) kit |
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- Not suitable for long-term DNA storage
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DNA extraction from amniotic fluid
| Method |
Protocols |
| Cell Lysate Preparation |
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| DNA extraction from amniotic fluid with magnetic beads |
Protocol available here |
| DNA extraction from amniotic fluid with QIAamp DNA Mini kit |
Protocol available here |
DNA extraction from Chorionic Villus Sampling (CVS)
| Method |
Protocols |
| Extraction from Chorionic Villus Sampling as described in Kooper et al., Detection of chromosome aneuploidies in chorionic villus samples by multiplex ligation-dependent probe amplification. J Mol Diagn; published online Dec 12, 2008 |
Protocol available here |
DNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissue
DNA extraction from Lithium Heparin blood samples
Many PCR reactions are sensitive to heparin, which may be carried over into DNA samples extracted from lithium heparin anticoagulated blood. The National Genetics Reference Laboratory in Wessex, UK, have followed the publication of Taylor 1997, which describes Heparinase I treatment of such samples, and have found it to be useful for cleaning up difficult samples prior to MLPA analysis. The protocol and more details can be found on their website: http://www.ngrl.co.uk/Wessex/mlpa.htm.
DNA extraction from fresh tissue
| Method |
Protocols |
| DNA extraction from fresh or frozen tissue |
Protocol available here |
RNA extraction from blood
| Method |
Advantages |
Disadvantages |
| Phenol/chloroform |
- Good DNA quality
- Inexpensive
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- Time-consuming
- Phenol may remain in sample
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RNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissue
4. Extra purification of contaminated DNA samples
| Method |
Advantages |
Disadvantages |
| Ethanol Precipitation (*) |
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| Phenol/chloroform |
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- Time-consuming
- Phenol may remain in sample
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| Silica-based columns (Qiagen, Promega) |
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(*) DNA losses can be reduced by inclusion of 10 μg glycogen (Roche Diagnostics, Cat. No. 10 901 393 001) in ethanol precipitations.
Last updated 19-01-2009
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