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Technical MLPA FAQ
To perform MLPA, is it necessary to get training?
Which controls are present in SALSA MLPA kits?
Which control samples should be included in the experiments?
Does MRC-Holland have positive controls available?
Is the purity and quality of the sample DNA important?
Is it possible to use DNA extracted from paraffin coupes for MLPA?
How important is the buffer that is used to dissolve the DNA?
Are MLPA reagents stable?
What are the unique features of the Ligase-65 enzyme?
Does Ligase-65 use ATP or NAD as a cofactor?
Do mismatches close to the ligation site of probes influence results?
Is it possible to compare results from different experiments?
Can I use a thermal cycler without heated lid?
Is it possible to use 0.5 ml tubes?
Why does the DNA detection/quantification protocol start with a 5 minute incubation at 98 °C?
Can I use smaller amounts of the probemix?
Can I decrease the duration of the hybridisation reaction?
Can I reduce the number of cycles in the PCR reaction?
Which factors influence the relative probe signals?
Is the MLPA generation of amplifiable probes compatible with amplification techniques other than PCR?
Does a touch-down PCR protocol improve results ?
Is it possible to use nested PCR techniques?
Can I get the exact sequences recognised by the SALSA probes ?
Is it possible for us to design a probe for a specific gene that is made of two synthetic oligonucleotides and include it in a mix?
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1. To perform MLPA, is it necessary to get training?
To perform MLPA, all you need is a thermocycler and a capillary sequence type electrophoresis. The MLPA detection/quantification protocol is quite simple and hence the MLPA technique is easily mastered as long as one has experience in working with these two instruments. Most laboratories implement the technique themselves and therefore we are of the opinion that MLPA training is not necessary. Any (technical) questions however can always be directed to: info@mlpa.com
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24. Is it possible for us to design a probe for a specific gene that is made of two synthetic oligonucleotides and include it in a mix?
Yes, we have successfully added completely synthetic probes generating amplification products with lengths of 100, 106, 112, 118 and 124 bp to our mixes. The guidelines for probe design are on our website. Most probe mixes contain a synthetic IL1B specific probe that results in a 94 bp amplification product. Do not add more than 2 femtomoles of each probe oligonucleotide in each MLPA reaction. It is also possible to order our DQ (DNA Quantification) and DD (DNA denaturation) control fragments, to include in your self-made probemix.
MRC-Holland has only used a very small number of synthetic probes. Although our experience with synthetic probes is very limited, our general impression is that the reproducibility of results is lower. Please tell us about your experiences.
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2. Which controls are present in SALSA MLPA kits?
Each SALSA MLPA probemix contains four DQ (DNA Quantity) control fragments. The amplification products generated by these DQ fragments are 64, 70, 76 and 82 bp long and thus much shorter than the amplification products generated by the probes. The purpose of these short DQ fragments is to give a clear warning signal when the amount of sample DNA used was lower than the 20 ng human DNA required for reliable MLPA results. Note that the amplification products of the DQ fragments are only visible when little or no DNA is present, and even when the ligation did not occur. Their peak size is inversely correlated with the amount of DNA present in the sample: when more than 100 ng of sample DNA is used, the four 64-82 bp DQ amplification products will be hardly visible.
The SALSA MLPA probemix also contains a 92 bp amplification product generated by an MLPA probe consisting of two synthetic probe oligonucleotides detecting a 2q14 DNA sequence. The peak area of this 92 bp fragment should be of similar size as most of the other MLPA amplification products. In contrast to the DQ control fragments, the 92 bp amplification product is sample DNA- and ligation-dependent and is thus only present when sufficient sample has been added and ligation occurred. The ratio between this 92 bp amplification product and the four DQ fragments can provide information over possible errors in the MLPA reaction.
In some MLPA kits (most are MS-MLPA kits) DNA denaturation (DD) control fragments are included. The DD control fragments consist of two synthetic probe oligonucleotides and their purpose is to give off a warning signal when the DNA has not been completely denatured. The peak size of the 92 bp amplification product in the DD control fragments reflects the copy number of a 2q14 DNA sequence. As said, the peak area of the 92 fragment should be of similar size as most of the other MLPA amplification products in the 130-454 bp range. Of the three DD control fragments, we deliberately added less of the 92 bp control probe, so that it becomes a measure for hybridisation. If the peak size of the 92 bp amplification product is much lower than that of the 88 and 96 bp fragments of the DD control mix, this may indicate that the hybridisation of probes to their targets has not been complete. Incomplete hybridisation can be the result of the use of insufficiently long hybridisation times; the use of insufficient amounts of probemix and/or MLPA buffer; the use of larger volumes of DNA solution (> 5 ul); a lower than 60 oC hybridisation temperature; or a lower than 105 oC temperature of the thermocycler lid.
The 88 bp DD control fragment is specific for a sequence in the CpG island preceding the FANCE gene on 6p21.3. The 96 bp DD control probe is specific for a sequence within the CpG island preceding the TP73 gene on 1p36. These two CpG islands both include a long sequence with a very high percentage of C/G nucleotides, and are therefore very difficult to denature. In case the 88 and 96 bp amplification products are much lower (less than 35% in peak area / peak height) as compared to the 92 and the 127-454 bp amplification products, denaturation of the sample DNA might have been incomplete, resulting in unreliable results for probes detecting sequences in or close (<5 Kb) to a CpG island. An extensive description of these control probes can be found in the MS-MLPA protocol.
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3. Which control samples should be included in the experiments?
This is a difficult question. For quality control of DNA specific probemixes, we compare MLPA results using different amounts of sample (0/20/80/240 ng human DNA) and, if applicable, male, female and/or trisomy DNA. MRC-Holland performs reactions on 50 ng human DNA using different hybridisation periods and different amounts of polymerase.
Many clients use 1-4 different DNA samples derived from healthy control persons and, if available, one or more samples with known deletions / duplications. For samples with a high chance of finding a copy number change such as DMD (P034-35) samples and CMT1 (P033) samples, it is essential to compare results on patients with samples from healthy controls. For many other applications, such as telomeric probemixes, it is sufficient in our view to test at least 8 patients in one experiment and to compare results obtained on a patient to the average of all results obtained. No need in our view in that case to include samples from healthy persons in the experiment for normalization of results.
For mRNA analysis, control reactions without addition of reverse transcriptase, reactions lacking sample RNA, and reactions on DNA samples, should yield almost identical results. The four small MLPA DQ control peaks (64, 70, 76 & 82 bp) should be very prominent in these samples.
No DNA control sample - Ligation-independent background
MLPA control reactions that completely lack sample DNA, or in which the Ligase-65 enzyme is omitted, should theoretically not yield any amplification products larger than 100 bp but are in practice often not completely blank. However, PCR is notorious to find always something to amplify. Compared to ordinary PCR reactions, MLPA is more prone to the generation of aspecific long amplification products in the absence of sample. Even without any ligation event, the M13 derived probe oligonucleotides are linearly amplified as the unlabeled PCR primer will bind to the long phosphorylated probe oligonucleotide and be elongated. In each PCR cycle, the complement will be made of almost all M13 derived probe oligonucleotides, resulting in an estimated 40 x 150.000.000 molecules in each PCR cycle, even when no sample DNA was present or no ligation occurred. If, after the first PCR cycle, the labeled PCR primer, or one of short probe oligonucleotides, inadvertently binds to the complement of one of the M13 derived probe oligonucleotides and is elongated by the polymerase, this elongation product contains both PCR primer sequences and will be exponentially amplified in the next PCR cycles. This can result in an amplification product longer than 100 nucleotides (Ligation-independent background). This is the reason why at least 20 ng human DNA should be used in MLPA reactions and this is also the reason why the "no dna blank" is not always completely clean. We compare the intensity of aspecific amplification products in the no dna control reactions with the intensity of the small control fragments at 64-70-76 and 82 nt. When the aspecific amplification products that are longer than 120 nt are less than 50% in peak area as compared to the four small control fragments of 64, 70, 76 and 82 nt, new lots will be approved. Please note that the aspecific amplification products in a "no dna" control reaction will be almost absent in MLPA reactions on > 20 ng sample DNA. Their relative peak height will decrease in samples with increasing amounts of sample DNA, similar to that of the 64-70-76-82 nt control fragments which are almost invisible in samples containing > 50 ng sample DNA.
No DNA control sample - Ligation-dependent background
A control reaction in which the sample DNA is omitted may yield a few ligation-dependent amplification products larger than 100 bp. In a typical MLPA reaction, approximately 20.000 copies of the human haploid genome are present as well as 600.000.000 molecules of each probe and 25.000.000.000 molecules M13 single stranded DNA. In a few rare cases we observed a limited amount of probe ligation without template, probably due to ligation of probe oligonucleotides while annealed to other probe molecules or to the M13 DNA. If no specific ligation products are present, even extremely small amounts of inadvertently ligated probes will give amplification products when using a PCR reaction with 30 or more cycles. This ligation-dependent background is higher when ligation reactions are allowed to proceed for more than the recommended 15 minutes, or are left at temperatures lower than 50 °C.
Contamination
If there appears to be a large number of >100 bp amplification products present in your sample, one of your reagents might be contaminated by amplification products of previous MLPA reactions. Be sure never to use micro-pipettes that have been used for handling MLPA PCR products when setting up a new MLPA reaction! PCR reaction vials should never be opened in the vicinity of the thermal cycler or the lab area where the MLPA reactions are prepared. As with other PCR reactions, it is necessary to exercise extreme care to prevent contamination in your lab.
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4. Does MRC-Holland have positive controls available?
No. Unfortunately, MRC-Holland has very limited access to patient samples.
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5. Is the purity and quality of the sample DNA important?
The purity is certainly important. The relative signal strength of some probes is dependent on the amount of polymerase activity during the PCR reaction. Impurities such as fenol or Trizol remnants can decrease the polymerase activity to such an extend that irreproducible MLPA results are obtained, even though ordinary PCR reactions are still possible. At very low polymerase activities, the relative intensities of all long fragments will be decreased compared to those of short fragments.
The quality of the DNA (average length) does not strongly influence relative probe signals. DNA samples that were degraded to such an extent that a 1000 bp PCR is no longer possible anymore will still yield excellent MLPA patterns, since the target sequence of SALSA MLPA probes is quite short (typically between 58-82 nt).
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6. Is it possible to use DNA extracted from paraffin coupes for MLPA?
Yes. On the support pages of our website www.mlpa.com, you can download a protocol for DNA extraction from formaldehyde-treated, paraffin-embedded tissues. The use of DNA extracted from paraffin coupes by other methods did not always result in satisfactory results.
DNA samples extracted from formalin-fixed, paraffin-embedded tissues often contain contaminants. We therefore recommend to compare these DNA samples with results obtained on DNA extracted by the same methods from paraffin-embedded healthy tissue. This way, it can be excluded that certain findings on an abnormal tissue sample are in fact due the effects of the formalin/paraffin treatment.
Formaldehyde cross-links should be destroyed by heat treatment; DNA should preferably be degraded to smaller fragments and be liberated from the tissue by protease treatment, before any tissue constituents are removed by centrifugation or phenol extraction.
Impurities in some DNA samples will decrease the effective polymerase activity during PCR. Reducing the amount of DNA used to 50 ng will sometimes have a beneficial effect.
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7. How important is the buffer that is used to dissolve the DNA?
The buffer used to dissolve DNA is important. The pH of the DNA preparation should be around 8.2 in order to prevent depurination during the initial heat treatment at 98°C. Some salts or other impurities in the sample might inhibit the ligase or polymerase enzymes. We recommend the use of DNA solutions in TE (10 mM Tris-HCl pH 8.2 + 0.1-1 mM EDTA). Do not use more than 1 mM EDTA in the sample solution!
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8. Are MLPA reagents stable?
Yes. Our results so far indicate that the probes, buffers and enzymes used are very stable. When testing product stability, we found that after incubation of all SALSA MLPA ingredients (probemix, buffers & enzymes) at room temperature for 11 weeks, good MLPA results were still obtained. All kit components are stable and will survive storage at room temperature for several weeks. Nevertheless, we recommend storage of Ligase-65 and SALSA-Polymerase at -20 °C. All other reagents can be left at 4 °C if used within 48 hrs. Otherwise they should be stored at -20 °C. Thawed reagents should always be briefly vortexed or mixed by "flipping" before use!!
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9. What are the unique features of the Ligase-65 enzyme?
Ligase-65 is active at 55-65 °C, in contrast to for instance T4-Ligase. Contrary to Taq-Ligase and Tth-Ligase, the Ligase-65 enzyme is easily inactivated before the start of the PCR reaction by briefly heating the MLPA reactions to 95-98 °C. Ligase-65 is very specific in that it will only ligate perfectly matched probe-target hybrids. In comparison to other ligases tested for MLPA reactions at MRC-Holland, Ligase-65 resulted in the most reproducible results and the lowest number of aspecific bands.
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10. Does Ligase-65 use ATP or NAD as a cofactor?
The cofactor used is NAD. It is present in Ligase buffer A.
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11. Do mismatches close to the ligation site of probes influence results?
Usually, a single mismatch will not influence hybridisation of the probe oligonucleotides to their targets. However, the ligation speed will be decreased by mismatches close to the ligation sites. The relative probe signal obtained will therefore depend on the duration of the ligation reaction and the amount of ligase used. Mismatches next to the ligation site even completely prevent formation of a probe signal under the MLPA experimental conditions. Mismatches within 3-4 bp of the ligation site will in general have a strong effect on the signal strength of that probe. Mismatches at other sites are expected to affect the signal strength only when the stability of the probe-target binding is severely decreased. When probes are designed, SNP's (Single Nucleotide Polymorphisms), as apparent from public databases such as Genbank, are avoided.
The amount of ligase used in combination with the ligation time used is 10-15 times the amount needed for ligation of 90% of the hybridized probes. As some probes ligate slower than others, an excess of ligase is needed.
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12. Is it possible to compare results from different experiments?
It is possible, but the best results are obtained when samples are compared that were analysed in the same experiment using the same Ligase-65/ligase buffer mix, the same polymerase master mix, the same PCR run and the same electrophoresis conditions. We recommend inclusion of a control reaction using standard DNA in each experiment.
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13. Can I use a thermal cycler without heated lid?
No. But a cheap clothes iron can replace the heated lid!
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14. Is it possible to use 0.5 ml tubes?
Yes, but due to the small volumes used, 200 µl tubes or microtiterplates are recommended.
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15. Why does the DNA detection/quantification protocol start with a 5 minute incubation at 98 °C?
The DNA detection/quantification protocol start with a 5 minute incubation at 98 °C both to denaturate the DNA and to generate fragments with an average size of 10 Kb. This will also reduce the viscosity of chromosomal DNA solutions. This heat treatment is not included in the mRNA analysis protocol as the cDNA is already small.
After addition of MLPA buffer and probes, samples are again heated for 1 minute at 95°C in order to reduce secondary structure of probes and dissolve probe aggregates.
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16. Can I use smaller amounts of the probemix?
This is not recommended. The probe mixes contain sufficient amounts of the probes to complete the hybridisation to target sequences within 6 hrs. We use a 16 hrs. hybridisation period in order to obtain the best reproducibility. The use of lower amounts of probes may result in an incomplete hybridisation of some probes.
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17. Can I decrease the duration of the hybridisation reaction?
Preliminary experiments indicate that shorter hybridisation periods are possible when the volume of the DNA sample is reduced from 5 to 2 µl.
Using more than the recommended amount of probes in order to decrease hybridisation times is not recommended, as it might result in a lower signal and increased background. The use of increased amounts of probe will result in a higher percentage of the unlabeled PCR primer being used for linear amplification of non-ligated probe molecules.
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18. Can I reduce the number of cycles in the PCR reaction?
Yes. The recommended PCR protocol (33 cycles using a 60 °C annealing temperature) is a compromise between optimum results and making the reactions foolproof. Reduction of the number of cycles to 30 is generally possible, but will usually not improve results. Relative peak areas of probe amplification products remain almost identical during additional PCR cycles (Nucleic Acid research (2003) 31, e153 supplementary material).
Multiplex PCR reactions using only one primer pair as used in AFLP, Differential Display and MLPA reactions slow down when the reactions run out of primers. This is in contrast to ordinary PCR reactions, in which amplification of the single amplification product slows down when reannealing of complementary strands is faster than annealing and elongation of primers.
AFLP and MLPA PCR reactions continue until the majority of the PCR primers have been consumed. The signals obtained will therefore be only slightly influenced by the number of extra cycles used or the amount of template used. The PCR protocol used is optimised for use with template amounts as low as 18 ng human chromosomal DNA, corresponding to 6000 copies of each single copy sequence.
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19. Which factors influence the relative probe signals?
The relative probe signal is the peak area of a probe amplification product, divided by the peak area of all probe amplification products. Probe design is a major factor influencing relative peak signals (Schouten,J.P. et al; NAR 30, e57). In addition, the quality of the probe oligonucleotides also has a strong effect. Long (>130) synthetic MLPA probes usually result in low peaks. Therefore, the SALSA MLPA probes made by MRC-Holland consist of one synthetic, relatively short fragment, and one longer, M13-derived fragment. These M13-derived long oligonucleotides are of much better quality than synthetic equivalents. Thirdly, the amount of KCl and Polymerase present during the PCR reaction can also influence relative probe signals.
In addition, there is also a relationship between relative probe signals and polymerase activity. The relative signal strength of some probes will decrease with reduced polymerase activity. It is therefore important that the polymerase master mix is well mixed before use. A major source of errors is incomplete mixing upon dilution of viscous (50 % glycerol) enzyme solutions. Paradoxally, a small number of probe signals will actually decrease with an increased polymerase activity. We have even noted some fragments that completely disappear with increased polymerase activity. For each newly-designed probe, we compare the results of PCR reactions in which a different amount of polymerase was used. Approximately 5% of newly-designed probes are replaced because a negative effect of polymerase activity on relative signal strength. We will thus always try to exclude such probes from MLPA kits.
Impurities in DNA samples will decrease the effective polymerase activity during PCR. Reducing the amount of DNA used will therefore sometimes have a beneficial effect. In particular, DNA samples extracted from formalin-fixed, paraffin-embedded tissues often contain contaminants. We therefore recommend comparing results of these DNA samples with results obtained from DNA extracted from paraffin-embedded healthy tissues that were treated identically.
The length of the amplification products had less influence on average probe signals (peak areas) when the PCR reactions where analysed on the Beckman CEQ capillary sequencer or on LICOR slab gels. However, when the ABI3700 capillary sequencer was used for analysis, the average probe signals of long amplification products were almost two times lower than for short products. This might be due to the injection procedure.
Relative probe signals are not strongly influenced by the amount of sample used, since the PCR slows down due to primer depletion. The number of PCR cycles has only a slight effect on the results obtained. We recommend to start with 33 PCR cycles. Usually this can be reduced to 30 cycles resulting in a slightly improved linear relation between target sequence copy number and relative probe signal. In case of low signals, an increase in the number of cycles to 36 will increase signal-noise ratio without affecting the relative probe signals.
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20. Is the MLPA generation of amplifiable probes compatible with amplification techniques other than PCR?
We did not try other amplification techniques than PCR. The stepwise amplification by PCR appears to be the most suitable method when reproducible relative quantification of probe-targets is the purpose.
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21. Does a touch-down PCR protocol improve results ?
No. Nice pictures can be obtained using other PCR protocols. However, relative peak areas of the probe amplification products change depending on the PCR protocol used. Even though each fragment is amplified with the same PCR primers, some fragments have stronger reduced amplification efficiencies at annealing temperatures in excess of 64°C. The annealing temperature and the speed of temperature changes are often not perfectly uniformly distributed accross the different parts of the thermocycler. The reproducibility of results may therefore be lower when the first PCR cycles have a low efficiency.
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22. Is it possible to use nested PCR techniques?
Our (limited) experience is that nested PCR can be used to increase sensitivity, but that the reproducibility of relative quantification will be lower. The labelled SALSA 582 PCR primer starts at the 5' end of the ligation products. Replacement of all short synthetic probe oligonucleotides by longer ones will provide the possibility to design a nested PCR primer. The unlabeled SALSA 577 PCR primer binds 14 nucleotides from the 3' end of the ligation products. The complete sequences common to all ligation fragments are in the extensive MLPA info on our web site.
Nested PCR techniques will not remove most ligation-dependent background. We are currently exploring other ways to perform MLPA reactions on sub nanogram amounts of DNA. If the sensitivity of the current MLPA protocol is insufficient for your application, please contact MRC-Holland.
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23. Can I get the exact sequences recognised by the SALSA probes ?
Yes. The Genbank sequence used and the ~20 nucleotides adjacent to the ligation site of the target sequence is in often described in the probemix description. The exact sequence recognised by a specific probe can be requested by sending an e-mail to info@mlpa.com. Please indicate the reason for the request
Please note that any information on SNP's/splice variants present in sequences detected by our SALSA MLPA probes is greatly appreciated.
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