MRC-Holland

MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

MLPA

MS-MLPA Two-Tube Protocol

Instructions For Use
MS-MLPA protocol for the detection and quantification of nucleic acid sequences and methylation
profiling.
To be used in combination with the appropriate MS-MLPA probe mix-specific product description.
MS-MLPA: Methylation-Specific Multiplex Ligation-dependent Probe Amplification.
NOTE: the standard protocol for MS-MLPA reactions is the MS-MLPA GENERAL protocol
(from here onwards called: the one-tube protocol). The MS-MLPA two-tube protocol
described here differs from the MS-MLPA one-tube protocol in that only part of the
ligation reaction is used for the PCR instead of the complete ligation reaction.
When to use the two-tube protocol:
The two-tube protocol may have advantages when using DNA samples containing impurities such
as high EDTA concentrations (>2.5 mM) or PCR inhibitors. Furthermore, using the two-tube protocol
makes it possible to repeat just the PCR using the remaining ligation reaction. This may be an
advantage when DNA is scarce and there is no possibility to repeat a failed run.
Cautions about the two-tube protocol:
Please take into account that the one-tube protocol is the general, improved MS-MLPA protocol
which is used to develop, optimise and QC test all MLPA probe mixes. MRC-Holland strongly
recommends using the one-tube protocol in consideration of the following: (1) compared to the twotube protocol, the probe standard deviation is slightly lower,(2) the risk of mixing up samples is
reduced, (3) less plastic is needed and (4) the one-tube protocol ensures easier handling.
Compared to the previous two-tube protocol using the older PCR primer mix (in use before June
2011), the following changes have been made to the two-tube protocol:
1.
2.
3.

The SALSA PCR buffer required for the two-tube protocol is no longer included in an MLPA reagent
kit. It can be ordered on special request free of charge.
The enzyme dilution buffer is no longer needed.
The polymerase mix for PCR reaction should be added at room temperature! Never use a hot start.

Manufactured by MRC-Holland B.V. Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands

Website : www.mlpa.com
E-mail : [email protected] (information & technical questions); [email protected] (for orders)
This protocol must be read in its entirety before using this product. For Research Use Only.
-------------------------------------------------------------------------------------------------------------------------------------------MS-MLPA Two-Tube Protocol Document History

Version MSTP-001 (09-08-2013)

-

NEW DOCUMENT. Fully reviewed MS-MLPA General Protocol completely adapted for use with the two-tube
method.

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

MLPA

Table of Contents
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

MS-MLPA ASSAY PRINCIPLE ...................................................................................................... 2

SALSA MS-MLPA ASSAY COMPONENTS & STORAGE CONDITIONS ............................................. 3
ASSAY SETUP INSTRUCTIONS ................................................................................................... 6
MS-MLPA REACTION - DNA DETECTION/QUANTIFICATION and METHYLATION PROFILING ............. 7
FRAGMENT SEPARATION BY CAPILLARY ELECTROPHORESIS (CE)................................................. 9
QUALITY CONTROL ................................................................................................................. 10
DATA ANALYSIS ...................................................................................................................... 13
INTERPRETATION OF RESULTS ................................................................................................ 15
MS-MLPA PROTOCOL FOR DNA ANALYSIS IN A NUTSHELL ........................................................ 17
EXPLANATION OF SYMBOLS USED ............................................................................................ 17

1. MS-MLPA ASSAY PRINCIPLE

Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) is used to determine the copy
number and methylation status of up to 50 DNA sequences in a single multiplex PCR-based reaction. Each MS-MLPA
reaction results in a set of unique PCR amplicons between 64-500 nt in length, which are separated by capillary
electrophoresis. In MLPA and MS-MLPA, not sample DNA is amplified during PCR, but MLPA probes that can hybridise
to the sample DNA. In contrast to a standard multiplex PCR, MS-MLPA uses a single PCR primer pair to amplify all
probes, making the method very robust.
The hybridising sequence of each probe is about 55-80 nt long. Each MLPA probe consists of two oligonucleotides that
must bind to adjacent target DNA sequences to be ligated. Upon ligation, a single molecule is formed that can be
amplified exponentially during PCR. Because hybridisation and ligation are essential for the probe to be amplified and
give a signal, unbound oligos do not give a signal and need not to be washed away. Following PCR, amplicons are
separated and quantified by capillary electrophoresis. Each probe in an MLPA probe set has a unique length and can
therefore be easily identified.
To determine both copy number and methylation status of the target DNA, each MS-MLPA probe set contains several
methylation-specific probes. These are designed to target DNA sequences which contain a cleavage site for the
methylation-sensitive restriction enzyme HhaI. After probe hybridisation, the MS-MLPA reaction is split into two parts.
One part of the MS-MLPA reaction is processed as a normal MLPA reaction, providing information on copy number
status of the target DNA (Error! Reference source not found.; 3A). The other part is treated with the HhaI
enzyme, which provides information on methylation status of the target DNA (Error! Reference source not found.;
3B).

Figure 1 MS-MLPA reaction.

When hybridising to an unmethylated DNA target, the methylation-specific probe will be ligated and simultaneously
digested by HhaI (Error! Reference source not found.; 3B bottom). A digested MS-MLPA probe will not generate a
peak signal because it cannot be amplified. In contrast, when the target sequence of the MS-MLPA probe is
methylated, the methyl group will prevent HhaI-digestion (Error! Reference source not found.; 3B top). An
undigested, ligated probe can be amplified during PCR, resulting in a normal peak signal.
MLPA is a relative technique: only relative differences can be detected by comparing the MLPA peak patterns of DNA
samples. Inclusion of reference samples in the same run is therefore essential. (See 7 DATA ANALYSIS.) Comparing
the electrophoresis patterns of the undigested MS-MLPA reactions allows for the detection of copy number changes
(Figure 2, top). Comparing the peak patterns of digested reactions may reveal unusual methylation of DNA target
sequences (Figure 2; bottom).

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

REFERENCE SAMPLE
Undigested reaction

MLPA

TUMOUR SAMPLE
Undigested reaction

Digested reaction

Digested reaction

Figure 2 Top: The tumour sample displays the same peak pattern as the normal reference sample, indicating there are no copy
number aberrations. Bottom: The digested reaction of the reference sample only shows peaks from probes lacking the HhaI
restriction site. For this application (ME001 probemix), all probe targets containing a HhaI-site are normally unmethylated and are
therefore digested. In contrast, in the digested tumour sample, extra peaks can be seen. Targets that are normally unmethylated
and therefore digested are methylated in this tumour sample.

Figure 3 Copy number changes as displayed by Coffalyser.Net software. Left: three probes in the test sample (bottom) are
found to have a reduced peak signal as compared to the reference sample (top). Right: Results after analysis by Coffalyser.Net:
arranging probes by chromosomal location shows a reduced copy number for the three adjacent probes.

Results of MS-MLPA tests depend on several factors, including sample purity, experimental setup (selection of test
and reference samples), instrument settings, data normalisation method, quality of the HhaI enzyme. For reliable MSMLPA results, the standard deviation for each MS-MLPA probe should be below 10% when testing normal DNA
samples. It is essential to follow the procedures described in this protocol accurately. Separate protocols exist for
standard MLPA (detection of copy number changes only) and RNA quantification (RT-MLPA).

2. SALSA MS-MLPA ASSAY COMPONENTS & STORAGE CONDITIONS

NOTE: MS-MLPA and standard MLPA make use of the same SALSA MLPA REAGENT KIT.
2.1.

REAGENT KIT ITEM NUMBERS

Item no.

Description

EK1-FAM
EK1-Cy5
EK5-FAM
EK5-Cy5
PCR001-FAM
PCR001-Cy5
PCR003-FAM

SALSA MLPA EK1 reagent kit 100 rxn SALSA MLPA EK1 reagent kit 100 rxn SALSA MLPA EK5 reagent kit 500 rxn SALSA MLPA EK5 reagent kit 500 rxn SALSA MLPA PCR kit 100 rxn - FAM
SALSA MLPA PCR kit 100 rxn - Cy5
SALSA MLPA PCR kit 300 rxn - FAM

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FAM
Cy5
FAM
Cy5

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

PCR003-Cy5

2.2.

SALSA MLPA PCR kit 300 rxn - Cy5

COMPONENTS PROVIDED PER SALSA MLPA REAGENT KIT1 (100 OR 500 REACTIONS)
Reagent kit
component
SALSA MLPA Buffer
(yellow cap) 2
SALSA Ligase-65
(green cap)
Ligase Buffer A
(transparent cap) 3
Ligase Buffer B
(white cap)
SALSA PCR Primer
Mix (brown cap)
SALSA Polymerase
(orange cap)

1
2
3
4
5
6

2.3.

Volumes
EK1

Ingredients

EK5

180 l 5180 l KCl, Tris-HCl, EDTA, PEG-6000, oligonucleotides. pH 8.5

Glycerol, BRIJ (0.05 %), EDTA, Beta-Mercaptoethanol (0.1 %), KCl, Tris-HCl. pH
7.5, Ligase-65 enzyme (bacterial origin)

115 l 5115 l

360 l 5360 l NAD (bacterial origin). pH 3.5

360 l 5360 l Tris-HCl, non-ionic detergents, MgCl2. pH 8.5
Synthetic oligonucleotides with fluorescent dye (FAM, Cy5, IRD800, ROX or
unlabeled), dNTPs, Tris-HCl, KCl, EDTA, BRIJ. pH 8
Glycerol, non-ionic detergents, EDTA, DTT (0.1 %), KCl, Tris-HCl, Polymerase
enzyme (bacterial origin). pH 7.5

240 l 5240 l
65 l

565 l

ADDITIONAL COMPONENT REQUIRED FOR TWO-TUBE PROTOCOL

Additional Component

Volume
4

SALSA PCR buffer SMR14 (red cap)

2.4.

Probemix (black cap)

Product Description

2
3

Tris-HCl, EDTA, KCl, non-ionic detergents. pH 8.5

Available Volumes

Ingredients

40 l (25R), 80 l (50R),
160 l (100R)
N/A

Synthetic oligonucleotides, oligonucleotides purified from

bacteria, Tris-HCl, EDTA. pH 8.0
Probemix-specific information

OPTIONAL: ADDITIONAL PCR REAGENT KIT (100 OR 300 reactions)

PCR kit component

1

480 l

Ingredients

APPLICATION-SPECIFIC MLPA PROBEMIX

Application-specific
MLPA probemix

2.5.

MLPA

SALSA PCR Primer

Mix (brown cap)
SALSA Polymerase
(orange cap)
Product Description

Volumes
PCR001

PCR003

240 l

3x 240 l

65 l

3x 65 l

N/A

N/A

Ingredients
Synthetic oligonucleotides with fluorescent dye (FAM, Cy5, IRD800, ROX or
unlabeled), dNTPs, Tris-HCl, KCl, EDTA, BRIJ. pH 8
Glycerol, non-ionic detergents, EDTA, DTT (0.1 %), KCl, Tris-HCl, Polymerase
enzyme (bacterial origin). pH 7.5
N/A

None of the ingredients are hazardous in the amount present in an MS-MLPA kit as defined in the Hazard
Communication Standard (HCS). None of the ingredients are of human or animal origin or are derived from
pathogenic bacteria. None of the preparations provided in this kit contain dangerous substances as defined in
European Directive 67/548/EC and its amendments at concentrations requiring distribution of Material Safety Data
Sheets as specified in European Directives 1999/45/EC and 2001/58/EC. Material Safety Data Sheets are not required
for these products.
2.6.

STORAGE AND SHELF LIFE

All components of the SALSA MS-MLPA kit must be stored directly upon arrival between -25C and -15C, shielded
from light and in the original packaging. When stored under the recommended conditions, a shelf life of at least 1
year is guaranteed, also after opening. See the labels on each vial for the exact expiry date.

1
2
3
4

For reverse transcriptase MLPA, separate reagent kits are available, see the General RT-MLPA protocol (EK1-RT, EK5-RT).
The improved MLPA buffer, introduced in January 2013, can be recognised by its yellow label.
The coenzyme NAD in ligase buffer A is sensitive to repeated freeze/thawing; it may deteriorate after more than 20 freeze/thaw cycles.
SALSA PCR buffer is required for this two-tube protocol and can be ordered free of charge on request.

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

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MLPA

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MRC-Holland
MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

2.7.

MATERIALS REQUIRED BUT NOT PROVIDED

2.8.

MLPA

HhaI restriction endonuclease. We recommend Promega R6441, 10 units/l.

Thermocycler with heated lid (99-105C)
Capillary Electrophoresis equipment (see section 5.2)
0.2 ml PCR tubes, strips or plates
Standard laboratory equipment

PRECAUTIONS AND WARNINGS

Always consult the most recent version of the relevant product description AND this general MS-MLPA
protocol for DNA detection/quantification and methylation profiling before use.
SALSA MS-MLPA kits are sold by MRC-Holland for research use only. These kits are not CE/FDA or
otherwise certified for use in diagnostic procedures.
For professional use only. Assay performance is dependent on operator proficiency and adherence to
procedural directions. Assay should be performed by professionals trained in molecular techniques. The
person responsible for result interpretation should be aware of the latest scientific knowledge of the
application in question and any limitations of the MLPA procedure that could lead to incorrect results.
SNPs/mutations within the sequence detected by a probe, as well as differences in purity between sample
DNA and reference DNA samples, can result in false positive results. All abnormalities detected by MLPA
should be confirmed by an independent technique, especially if they concern a single MLPA probe.

3. ASSAY SETUP INSTRUCTIONS

3.1.

SAMPLE TREATMENT
1. Use a total quantity of 50-250 ng (optimum: 50-100 ng) of human DNA in a 5 l 5 volume for each MLPA
reaction6. If necessary, DNA samples can be concentrated by ethanol precipitation. (Glycogen (Roche
901393) can be used as carrier.) 7
2. Dissolve and dilute sample DNA in TE (10 mM Tris-HCl pH 8.2 + 0.1 mM EDTA). DNA preparation should
have pH: 8.0-8.5 to prevent depurination during initial heat treatment at 98o C. If unknown whether
sufficient TE is present in the sample, add Tris-HCl: 4l sample DNA + 1l 50mM Tris-HCl pH 8.5
3. Extraction methods should not leave a high concentration of contaminants such as salt. Do not use Qiagen
EZ1, M6, M48 and M96 systems, unless an alternative protocol is used.
4. We have tested and can recommend the following extraction methods:


Qiagen Autopure LS (automated) and QIAamp DNA mini/midi/maxi kit (manual)


Promega DNA extraction Wizard (manual)


Salting out (manual)
5. MLPA is more sensitive to contaminants than simple monoplex PCR assays. Contaminants include
(guanidinium) salts, phenol, ethanol, heparin, EDTA, Fe. To minimise the effect of contaminants, ensure
extraction methods, tissues types, concentrations and treatment are as similar as possible.
6. EDTA concentration of the samples should not be higher than 1.5 mM. Sample DNA should not be
concentrated by evaporation or SpeedVac as this leads to a very high EDTA concentration.
7. In case of doubts about DNA quality: a) use only 20-50 ng of sample DNA; b) clean contaminated samples
by ethanol precipitation or silica based clean-up kits.
8. Do not use heparinised blood for DNA extraction. Traces of heparin are very difficult to remove from DNA
preparations and can distort the MS-MLPA PCR reaction.
9. An RNAse treatment is essential when investigating genes that are highly expressed in the sample tissue
studied. E.g. HBA, HBB genes in blood-derived samples; (mitochondrial) ribosomal RNA genes (all tissues).

Never use more than 5 l sample DNA per reaction. Using more than 5 l DNA reduces the probe and salt concentration. This reduces the
hybridisation speed and the stability of the binding of MLPA probes to the sample DNA.
6
Optical density (260 nm) measurements often overestimate the DNA concentration e.g. due to contamination with RNA. Whether the DNA
quantity was sufficient can be estimated on the basis of the Q-fragments, as is explained in 6.3.
7
DNA from whole genome amplification is not recommended for MLPA because of the possible amplification bias among the target sequences.

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

3.2.

MLPA

SELECTING REFERENCE & OTHER CONTROL SAMPLES

1. REFERENCE SAMPLES. Minor differences between the test procedure may affect the MS-MLPA peak pattern.
Therefore, reference samples should be included in each MS-MLPA run! Always compare samples that have
been a) run within the same test run; b) tested with the same probemix lot.
2. MULTIPLE REFERENCE SAMPLES are needed to estimate the reproducibility of each probe within each MSMLPA run. Use at least 3 different reference samples per MS-MLPA run. When using more than 21 samples,
add 1 additional reference sample for every 7 additional samples. Reference samples should be distributed
randomly over the sample plate to minimise variation.
3. SELECTING REFERENCE SAMPLES. Reference samples are DNA samples in which the target and reference
probe sequences are assumed to have a normal copy number. They are usually obtained from healthy
individuals. They should be as similar as possible to the test samples in all other aspects (see 3.1 SAMPLE
TREATMENT). For formalin-fixed paraffin-embedded (FFPE) tissue, use reference samples derived from
similarly treated, healthy tissue if possible. When selecting reference samples, please note that methylation
patterns may vary between tissues and even age groups! A sequence that is always methylated in bloodderived DNA may be unmethylated e.g. in DNA from amniotic fluid.
4. COMMERCIAL DNA. In case of doubts about sample quality, it is advised to include one or more commercial
DNA samples. We recommend Promega Cat. Nr G1471 male & G1521 female DNA.
5. NO DNA CONTROL. Per MS-MLPA run, include a no DNA control reaction in which DNA has been replaced by
TE (10 mM Tris-HCl pH 8.2 + 0.1 mM EDTA). This may reveal possible contamination of TE, MLPA reagents,
electrophoresis reagents or capillaries.
6. POSITIVE CONTROL SAMPLES. Inclusion of positive control samples is recommended when available. When
using DNA from cell lines as positive control sample, please note that cell lines may have acquired additional
copy number changes, including gains or losses of complete chromosomes.
7. ALIQUOT PRECIOUS REFERENCE/CONTROL SAMPLES and store these at -20o C. Contamination with
microorganisms or moulds can deteriorate samples that are stored at 4oC for an extended period.

4. MS-MLPA REACTION - DNA DETECTION/QUANTIFICATION and METHYLATION PROFILING

4.1.

NOTES TO READ BEFORE YOU START (DAY 1)

The SALSA PCR buffer is not included in an MLPA reagent kit but required for this (two-tube) protocol. It
can be ordered free of charge.
The enzyme dilution buffer (in use before 2012) is no longer needed.
Use a thermocycler with heated lid (99-105C).
After use, store all reagents between -25C and -15C.
Always vortex thawed buffers and probemix before use, as ingredients concentrate at the bottom of the
tubes. MLPA buffer is typically frozen at -20o C but may remain fluid due to high salt concentration.
Centrifuge all MLPA reagent tubes for a few seconds before use, as drops may have adhered to the lid.
Enzyme solutions contain 50% glycerol and remain fluid at the recommended storage temperature. Never
vortex enzyme solutions. Master mixes containing enzymes should be mixed by gently pipetting up and
down. When the viscous enzyme solution is not mixed properly with the buffers, unreliable results will
occur! If the enzyme solution is mixed too vigorously however, enzyme inactivation occurs. When preparing
master mixes, always add enzymes last.
To minimise sample variation, prepare sufficiently large volumes of master mix solutions. Include a 5-10%
volume surplus to allow for pipetting errors. Prepare Ligase-65 master mix and Polymerase master mix < 1
hour before use and store on ice.
When running a large number of samples, use multi-channel pipettes. The ligation incubation period of the
first tubes will be longer when running many samples, but this does not influence results.
Compared to the general MS-MLPA protocol (the one-tube protocol), in the two-tube protocol only part (10
l) of the ligation reaction is used for the PCR instead of the complete ligation reaction. The two-tube
protocol may have advantages when using DNA samples containing impurities such as high EDTA
concentrations (>1.5 mM) or PCR inhibitors.

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

4.2.

MLPA

THERMOCYCLER PROGRAM FOR THE MS-MLPA REACTION

1) DNA denaturation
1.
98C
2.
25C

5 minutes
pause

2) Hybridisation
3.
4.
5.

1 minute
pause
pause

reaction
95C
60C
20C

3) Ligation & Ligation-Digestion reactions

6.
48C
pause
7.
48C
30 minutes
8.
98C
5 minutes
9.
20C
pause
4) PCR reaction
10.

11.
12.
4.3.

35 cycles:

72C
15C

95C
60C
72C

30 seconds
30 seconds
60 seconds

20 minutes
pause

DNA DENATURATION (DAY 1)

1. Label 0.2 ml tubes, strips or plates.
2. Add 5 l DNA sample (50-250 ng; 50-100 ng is optimal) to each tube. Use TE for no DNA control.
3. Place tubes in thermocycler; start MLPA thermocycler program (see above). Denature sample DNA for 5 min
at 98C; cool samples to 25C before removing tubes from thermocycler.

4.4.

HYBRIDISATION REACTION (DAY 1)

1. Vortex viscous MLPA buffer and MLPA probemix before use.
2. Prepare hybridisation master mix containing, for each reaction: 1.5 l MLPA buffer (yellow cap) + 1.5 l
probemix (black cap). Mix hybridisation master mix well by pipetting or vortexing.
3. After DNA denaturation, add 3 l hybridisation master mix to each sample tube. Mix well by pipetting up and
down.
4. Continue thermocycler program: incubate for 1 min at 95C, then 1620 hours at 60C.

4.5.

LIGATION & LIGATION-DIGESTION REACTION (DAY 2)

1. Vortex the two Ligase Buffers before use.
2. Prepare a Ligase-65 master mix. For each reaction, mix: 8.25 l dH2O + 1.5 l Ligase buffer B (white cap) +
0.25 l Ligase-65 enzyme (green cap). Mix well by pipetting gently up and down. Never vortex enzyme
solutions.
3. Prepare a Ligase-Digestion master mix. For each reaction, add: 7.75 l dH2O + 1.5 l Ligase buffer B (white
cap). Then add 0.25 l Ligase-65 enzyme (green cap) and 0.5 l HhaI enzyme (Promega R6441, 10 units /
l). Mix well by pipetting gently up and down.
4. Continue thermocycler program: pause at 20C. Remove the tubes from the thermocycler.
5. Add 3 l Ligase buffer A and 10 l water to each tube. Mix by gently pipetting up and down. Separate the
mixture by transferring 10 l of the whole mixture to a second tube.
6. Place the tubes in the thermocycler. Continue the thermocycler program: pause at 48C.
7. When the thermocycler is at 48C, add 10 l of the Ligase-65 master mix to the first tube (copy number
test). Mix by gently pipetting up and down.
8. Add 10 l of the Ligase-Digestion mix to the second tube (methylation test). Mix by gently pipetting up and
down.
9. Continue thermocycler program: 30 min incubation at 48C (for ligation and HhaI digestion); 5 min at 98C
for heat inactivation of the enzymes. Pause at 20C. At this point, remove tubes from thermocycler.

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MS-MLPA DNA protocol version MSTP-v001; last update 09-08-2013

4.6.

MLPA

PCR REACTION (DAY 2)

Note: as compared to the PCR reaction in standard MLPA protocol, the PCR protocol for MS-MLPA described here
makes use of half volumes. Some users prefer doing the PCR using double these volumes. The optional SALSA PCR
reagents (PCR-001) can be ordered for this purpose.
1. Label 0.2 ml tubes, strips or plates of PCR reaction.
2. Prepare a PCR buffer mix containing for each reaction: 4 l SALSA PCR buffer + 26 l dH2O. Mix briefly by
vortexing.
3. Add 30 l of the PCR buffer mix to each new tube.
4. At room temperature, transfer 10 l of each ligation product to its corresponding PCR tube.
5. Vortex SALSA PCR primer mix. Warm polymerase for 10 sec in your hand to reduce viscosity.
6. Prepare polymerase master mix by adding, for each reaction: 3.75 l dH2O + 1 l SALSA PCR primer mix
(brown cap) + 0.25 l SALSA Polymerase (orange cap). Mix well by pipetting up and down; do not vortex.
Store on ice until use.
7. At room temperature8, add 5 l polymerase mix to each tube. Mix by pipetting gently up and down.
Continue thermocycler program; 35 cycles of 30 seconds 95C; 30 seconds 60C; 60 seconds 72C. End
with 20 min incubation at 72C; pause at 15C.
8. After PCR reaction, do not open tubes in the room with the thermocycler. To avoid contamination, use
different micropipettes for performing MLPA reactions and handling MLPA PCR products.
9. PCR product can be stored at 4C for 1 week. For longer periods, store between -25C /-15C. As
fluorescent dyes are light-sensitive, store PCR products in dark box or wrapped in aluminium foil.

5. FRAGMENT SEPARATION BY CAPILLARY ELECTROPHORESIS (CE)

5.1.

NOTES TO READ BEFORE YOU START

Size standard, run conditions, polymer, fluorescent dye and volume of MLPA PCR reaction depend on CE
instrument type. Settings below are standard settings. Instrument settings may require optimisation for
optimal MLPA fragment separation; follow instructions of your CE supplier.

Using old capillaries or polymer has a detrimental effect on MLPA results. Change capillaries and polymer
regularly. Polymer quickly deteriorates after prolonged exposure to >25oC. When not using instrument daily,
remove polymer after use and store at 4o C. In case size standard peaks are low and broad, the capillaries or
polymer are usually deteriorated.

Formamide can become acidic when stored for longer periods. This can result in depurination and
fragmentation of DNA upon heating. Store formamide in aliquots at -20o C.

In case all MLPA peaks are low, do not add more MLPA PCR product to injection mixture! Adding more PCR
product increases the salt concentration in the injection mixture, competing with the DNA for injection.
When an increase in peak heights is desired, increase injection time or voltage instead.

Reset the bin settings in your fragment analysis software when using a different MLPA probemix lot, size
standard, CE instrument or run settings.

5.2.

EQUIPMENT AND REAGENTS REQUIRED BUT NOT PROVIDED

1. Capillary electrophoresis instrument with fragment analysis software.
a. Beckman: GeXP Software Package
b. Applied Biosystems: Standard Foundation Data Collection Software.
2. High quality formamide (e.g. Hi-Di Formamide, Applied Biosystems 4311320).
3. Labelled size standard.
a. Beckman: CEQ DNA Size Standard Kit - 600 (p/n 608095)
b. Applied Biosystems: GeneScan 500, 600 LIZ (preferred); 500 ROX, 500 TAMRA.
4. Polymers:
a. Beckman: GenomeLab Linear Polyacrylamide (LPA) denaturing gel 391438.
b. Applied Biosystems sequencers - preferred: POP-4 or POP-7; POP-6 is not recommended because of
its high resolution. For some older sequencers, POP-6 may be the only option.

8
With the introduction of the new PCR primers (recognisable by the label with MRC-Holland logo) in June 2011, the temperature at which the PCR
should be prepared has been changed to Room Temperature (~20C).

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5.3.

MLPA

ELECTROPHORESIS SPECIFICATIONS

Instrument

MLPA
Primer
Dye

Capillaries

Injection mixture

Initial Settings

Beckman
CEQ-2000
CEQ-8000
CEQ-8800

Cy5

33 cm

0.7 l PCR reaction

0.2 l CEQ - size standard 600
32 l HiDi formamide / Beckman SLS
Add one drop of high quality mineral oil.

ABI-Prism 3100 (Avant)

ABI-3130 (XL)
ABI-3500
ABI-3730 (XL)

FAM

36, 50 cm

0.7 l PCR reaction

0.3 l ROX / 0.2 l LIZ size standard
9 l HiDi formamide
Seal the injection plate, incubate for 3 min
at 86C, cool for 2 min at 4C. 9

ABI-Prism 310

FAM

run method: Frag

capillary temperature: 50C
denaturation: 90C for 120 sec
injection voltage: 1.6 kV
injection time: 30 sec
run time: 60 min
run voltage: 4.8 kV
run module: FragmentAnalysis
(compatible with array length)
injection voltage: 1.6 kV
injection time: 15 sec
run voltage: 15 kV
run time: 1800 s
oven temperature: 60C
injection voltage: 1.6 kV
injection time: 15 sec
filter set: D
polymer: POP4

ABI-3500:
50 cm!

47 cm

0.75 l PCR reaction

0.75 l dH2O
0.5 l size standard
13.5 l HiDi formamide
Incubate mix for 3 min at 86C, cool
for 2 min at 4C. 8

6. MLPA QUALITY CONTROL

MLPA Internal Quality Control Fragments must be checked to ensure minimal quality requirements are
met! MS-MLPA probe sets contain quality control fragments that signal problems that may affect MS-MLPA results.
Inspect the quality of the MS-MLPA, including quality control fragments using the flowchart below. Only data that
meets the quality requirements is suitable for MS-MLPA result calculation. If needed, repeat capillary electrophoresis
or MS-MLPA reaction.
6.1.

DATA VISUALIZATION
1. Visualizing your raw data (.fsa, .scf, .cqf, .esd files prior to size-calling):
a. Beckman: Coffalyser.Net or Beckman GenomeLab software.
b. ABI: Coffalyser.Net, GeneMapper, GeneMarker, Peak Scanner or Foundation Data Collection Software on
CE instrument.
2. Size-calling your data:
a. Beckman: use Coffalyser.Net or CEQ / GeXP GenomeLab software to size-call and visualise data.
b. ABI: use Coffalyser.Net, GeneMapper, GeneMarker or Peak Scanner or to size-call and then visualise
data.

6.2.

MS-MLPA QUALITY CONTROL FRAGMENTS

Table 1 Internal quality control fragments

Name
Length (nt)
Interpretation
92 nt benchmark
Normal probe; is a benchmark to compare other quality control fragments
92
probe
to.
High when DNA amount is too low or ligation failed. All four Q-fragment
Q-fragments
64, 70, 76, 82
signals > 33% of the 92 nt control fragment  DNA quantity insufficient.
Low in case of poor sample DNA denaturation. Signal < than 40 % of the
D-fragments
88, 96
92 nt control fragment  DNA denaturation problems.
X & Y fragments
100, 105
Control for sample swapping.10

Briefly heating the injection mixture before capillary electrophoresis is essential. If this is omitted, some sequences with a high Guanine and
Cytosine content may remain double-stranded, thereby altering their mobility.
10
Rare cases are known in which this Y-specific sequence is absent in males, as well as females who carry this Y-sequence on an X-chromosome.

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Q-FRAGMENTS
The 4 Quantity Fragments (Q-fragments, at 64-82 nt) measure whether sufficient DNA was added and whether
ligation was successful. The Q-fragments do not need to hybridise to the DNA or be ligated to be amplified during
PCR. The more sample DNA is added, the lower they get; see Figure .


ALL FOUR Q-FRAGMENTS HIGH: INSUFFICIENT SAMPLE DNA PRESENT OR LIGATION REACTION
FAILED!

Figure 4 Effect of DNA quantity on Q-fragments. The more sample DNA is used, the lower the Q-fragments. Shown are
MLPA results with A. 5 ng DNA, B. 10 ng, C. 50 ng.

D-FRAGMENTS
The 2 DNA Denaturation Fragments (D-fragments, at 88, 96 nt) detect a sequence in a strong CpG island. CpG islands
have a high CG content and are difficult to denature. They are low in case of denaturation problems.
When using ABI POP7, a non-specific fragment of ~87 nt is usually present and may coincide with the 88 nt Dfragment. Please note that only the appearance of all three peaks at 88, 92 and 96 nt indicates DNA contamination of
the RNA sample.


D-FRAGMENTS LOW: SAMPLE DNA DENATURATION INCOMPLETE!

Figure 5 Effect of poor denaturation on D-fragments. D-fragments are low when sample DNA denaturation is incomplete
(here induced by adding salt). MLPA results on DNA sample containing in A. TE; B. TE + 40 mM NaCl; C. TE + 100 mM NaCl. As
MS-MLPA probes more frequently target CG-rich sequences, denaturation problems may be more prevalent in MS-MLPA.

6.3.

NO DNA REACTION

In a typical No DNA control, only the four Q-fragments are visible. However, MLPA PCR reactions are more prone to
non-specific peaks in the No DNA than a normal PCR reaction. In some probe sets, a few peaks may be visible. These
non-specific peaks should not influence MLPA results: when sufficient sample DNA is used, they are outcompeted by
the MLPA probes. Notify MRC-Holland in case a non-specific peak in the No DNA reactions is reproducibly higher than
the Q-fragments.
6.4.

EVAPORATION PROBLEMS

Evaporation occurs during (A) pipetting the ligation reaction at 54C or (B) overnight hybridization. In case you
suspect evaporation problems (see flowchart), the following may help. (A): Reduce handling time by using multichannel pipettes. (B): Test evaporation by incubating 8 l H2O overnight at 60oC; >5 l H2O should remain. To
reduce evaporation: 1. ensure heated lid works well; 2. increase/decrease pressure of lid on tubes; 3. try different
tubes (ABgene AB-0773, AB-0451); 4. use mineral oil (Vapor-lock, Qiagen): add small drop of oil to DNA sample, just
enough to cover it. There is no need to remove oil. After adding MLPA buffer-probemix mixture and polymerase mix:
centrifuge very briefly. After addition of ligase mix: gently pipet up and down.
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6.5.

MLPA

QUALITY CONTROL FLOWCHART

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7. DATA ANALYSIS
Only data that has passed quality evaluation can be used for data analysis. We recommend Coffalyser.Net for MSMLPA data analysis as it has been designed by MRC-Holland as the optimal data analysis program, with a thorough
analysis algorithm and built-in quality checks. The method below describes the very basic principles of MLPA data
normalisation. For certain applications, a more thorough analysis may be required, see the probemix-specific product
description). The analysis of MS-MLPA probemixes consists of two parts:
7.1 PART 1: DETERMINING COPY NUMBERS: determine copy numbers by comparing undigested samples.
This is identical to standard MLPA and involves two steps. Note that the method described here makes use
of a normalisation constant, which offers a more thorough analysis.
7.2 PART 2: DETERMINING METHYLATION PROFILE: determine methylation patterns by comparing each
undigested sample to its digested counterpart. The second part is unique for MS-MLPA probemixes and
serves to quantify the percentage of methylation within a given sample.
7.1.

PART 1: DETERMINING COPY NUMBERS

The absolute fluorescence detected by the CE instrument depends on many factors and needs to be normalised to
obtain useful MS-MLPA results. Part 1, MLPA copy number analysis, involves two normalisation steps: one within the
sample (intrasample normalisation) and one between samples (intersample normalisation).
1. Intrasample normalisation: within each (digested and undigested) sample, compare the probe peaks
detecting the target genes TO reference probe peaks. (Reference probes detect sequences that are
expected to have a normal copy number in all samples; they do not contain a site for the HhaI enzyme).
This should be done by dividing the signal of each probe by the signal of every reference probe in that
sample, thus creating as many ratios per probe as there are reference probes. Subsequently, the median of
all these produced ratios per probe should be taken; this is the probes Normalisation Constant. This
Normalization Constant (NC), can then be used for intersample normalisation (step 2 below) and for
determining the methylation profile (7.2 PART 2: DETERMINING METHYLATION PROFILE)
2. Intersample normalisation: compare peak pattern of DNA sample of interest TO reference DNA samples.
(Reference DNA samples are derived from healthy individuals, expected to have a normal copy number and
methylation status This way, the final probe ratio for a probe is calculated, which is called the Dosage
Quotient (DQ):for the regions of interest.)
DQ probe n =

[NC for probe n in undigested test sample A]

--------------------------------------------------------------------------------[average NC for probe n in the undigested reference samples X, Y, Z]

An abnormal DQ may indicate a deletion or duplication of MLPA probe targets, as shown in Figure 6. Arranging probes
according to chromosomal location facilitates the interpretation of results and may reveal more subtle changes such
as mosaicism.

Figure 6 Data analysis of the undigested reactions, providing information on copy number status. Shown is the ME028-A1
PWS/AS probemix that contains probes for the imprinted 15q11-q13 region. Left: 22 probes in the undigested test sample
(bottom) show reduced peaks (arrows) as compared to the undigested reference sample (top). Right: normalised data shows that
the 22 affected probes in the test sample (dots) form a contiguous region with a significantly lower copy number as compared to
the reference samples (boxplots), implying one copy of the region is deleted.
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For a reliable analysis, the DQ results should meet two criteria:

1) Reference Samples (undigested): the standard deviation of all probes should be 0.1 and DQ between
0.85-1.15
2) Test Samples (undigested): the standard deviation of the reference probes should be 0.10 and DQ
between 0.85-1.15
When probes do not meet these criteria, re-examine the raw data. When probes do meet these criteria, the dosage
quotients can be used as an indication to identify abnormal copy numbers:
Table 2 Dosage Quotients
Copy Number Status
Normal
Heterozygous duplication
Triplication
Heterozygous deletion
Homozygous deletion
Ambiguous copy number

Dosage Quotient
0.85 < DQ < 1.15
1.35 < DQ < 1.55
1.70 < DQ < 2.20
0.35 < DQ < 0.65
0
All other values

These values are estimates. To reliably determine copy numbers, the exact cut-off values for the dosage quotients will
differ for each probe as they depend on the standard deviation of that probe. The standard deviation is affected by
DNA sample quality, selection of reference DNA samples and quality of the MLPA reaction and capillary
electrophoresis. Coffalyser.NET MLPA software is available to calculate dosage quotients taking the probe standard
deviation into account. Other software and self-made analysis sheets may be less reliable. Validate any analysis tools
prior to use by analysing known samples. Always visually inspect each MLPA reaction first, following the guidelines in
the previous chapter.
7.2.

PART 2: DETERMINING METHYLATION PROFILE

Determine the methylation status of each MS-MLPA probe: compare the peak pattern of each digested DNA sample
TO the corresponding undigested sample. The approximate methylation percentage per probe is calculated as
follows:
[NC for MS-MLPA probe n in digested test sample A]
% methylation =
------------------------------------------------------------------ * 100%
[NC for MS-MLPA probe n in undigested test sample A]
The methylation profile of a test sample is assessed by comparing the probe methylation percentages obtained on the
test sample to the percentages of the reference samples (Figure 7). (Reference DNA samples are derived from
healthy individuals, expected to have a normal copy number and methylation status for the regions of interest.) This
way, differences in methylation status between the sample of interest and the reference samples can be identified.

Figure 7 Profile comparison of MS-MLPA data. Left: reference sample; undigested (top) and digested (bottom). Right: test
sample; undigested (top) and digested (bottom). The arrows represent the calculations that are made: horizontal arrow copy
number quantification using the undigested reaction of the reference sample and the test sample; vertical arrows methylation %
determination using the digested and undigested reaction of a sample. The methylation profile of a sample is judged by comparing
its methylation percentages to those of the reference samples (not represented by an arrow as it is not a calculation).

For a reliable methylation analysis, two extra criteria apply:

All samples (digested): The probe signal of the Digestion Control Probes should be 4% or less of the
corresponding probe signals in the undigested reactions.
All samples (digested): The dosage quotient of the reference probes, obtained in step 3 of the data
analysis, should be between 0.7 and 1.3.
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Figure 3 Electropherograms (detail) of three samples analysed with

ME028-A1 Prader-Willi/Angelman Syndrome (above: undigested, below:
digested) and a ratio chart showing MS-MLPA results generated with
Coffalyser.Net.
The three samples have identical copy numbers, but differ in methylation
status of the target of probe 5.
Middle - In normal individuals, the paternal copy of the gene detected by
probe 5 is unmethylated, whereas the maternal copy is methylated. This is
illustrated by the reference sample (middle), where the probe 5 peak in the
digested sample is halved as compared to the undigested sample, indicating
50% methylation is the norm.
Left PWS disomy: digested sample shows a probe 5 peak that is just as
high as in the corresponding undigested sample, indicating 100%
methylation of the target DNA. Since the copy number in this individual is
normal (2) but both copies are found to be methylated, this will result in
PWS. The most common cause of this finding is a maternal disomy, in which
two imprinted copies are present on the maternal X-chromosome (and none
on the paternal one). Another, less common cause for PWS is an imprinting
defect: the paternal X-chromosome carries a wrong, maternal methylation
imprint and is therefore methylated. Since MLPA cannot localise the copies
present, these two causes cannot be distinguished.
Right AS disomy: digested sample shows a complete absence of probe 5,
indicating 0% methylation of the target DNA. Since the copy number in this
individual is normal (2) but both copies are found to be unmethylated, this
would result in Angelman syndrome. The most common cause of this finding
is a paternal disomy, in which two non-imprinted copies were present on the
paternal chromosome (and none on the maternal one). Another, less
common cause for AS is an imprinting defect: the maternal copy carries a
wrong paternal methylation imprint and is therefore unmethylated.
Note: probe 7 detects a sequence that is normally unmethylated in bloodderived DNA; this probe is used to verify that HhaI-digestion was complete.
The ratio chart shows the dosage quotients of a digested sample. The boxplots indicate the reference sample population: the
target DNA of 4 probes is 50% methylated compared to the 100% methylation of the other probes target DNA.

8. INTERPRETATION OF RESULTS
To judge whether the results obtained are reliable, a good understanding of the MS-MLPA technique and the
application screened for is essential. Keep the following in mind:

MS-MLPA probe sets cannot detect deletions/duplications lying outside the target sequence of its probes. MSMLPA probes typically detect a sequence of ~55-80 nt. Most product descriptions mention only partial probe
sequences. Complete probe sequences are available on www.mlpa.com or via [email protected].
Sequence changes (SNPs, point mutations) in the probes target DNA sequence (even when not in the direct
vicinity of the ligation site) can cause false deletions. SNPs can reduce the probe signal by preventing ligation or
by destabilising the binding of the probe oligonucleotides to the sample DNA11. Confirm (especially smaller)
deletions by sequencing. Frequency of single exon deletions is generally higher in genes with large introns.
Copy number changes detected by a single probe always require confirmation by other methods. Sequencing is
often used to determine whether a mutation or polymorphism affecting the probe signal is present in the probe
target sequence. Long-range PCR and qPCR are often used to confirm (single) exon deletions. It is possible to
design your own synthetic MLPA probes for confirmation of results (e.g. Hills A. et al., (2010) Mol Cytogenet.
3:19). Information on MLPA probe design is available on www.mlpa.com.
Not all deletions and duplications detected by MLPA are pathogenic.
For many genes, exons are described that are only present in certain transcript variants. MRC-Holland cannot
provide information whether or not deletion or duplication of such exons will result in disease. For some genes,

11
When designing probes, known SNPs are avoided when possible. However, new SNPs are continuously being discovered. Please notify us when a
polymorphism or a frequent pathogenic mutation influences a probe signal.

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in-frame deletions resulting in mild, or no disease, have been described. A duplication of one or more exons may
disrupt that copy of the gene resulting in disease, whereas a complete gene duplication may not be pathogenic.
Germline copy number variations reported in healthy individuals can be found in http://projects.tcag.ca/variation/.
Certain copy number aberrations can be due to somatic alterations. For instance, a somatic trisomy 12 is an early
sign of Chronic Lymphocytic Leukemia (CLL).
For most applications, the major cause of genetic defects will be small (point) mutations, most of which will not
be detected by SALSA MLPA kits. MLPA will not detect most inversions, translocations, nor copy number changes
that lie outside the sequence detected by the MLPA probes.
In case of poor sample DNA denaturation, even the apparent deletion of several probes recognising adjacent
genomic targets can be a false positive result! The presence of salt in DNA samples (e.g. 40 mM NaCl or 1 mM
MgCl2) prevents DNA denaturation of CG-rich chromosomal regions. Sequences in the vicinity of such CpG islands
will denature at 98o C but will reanneal immediately upon cooling as the non-denatured CpG island holds the two
strands together. Binding of certain probes to their target sequence will be hindered, resulting in reduced signals
for probes located within several kb from such strong CpG islands. This is why the D-fragments should always be
examined with great care.
MLPA tests provide the average copy number of the target sequences in the cells from which the DNA sample was
extracted. In case several probes targeting adjacent sequences have an unusual value but do not reach the usual
threshold values for a deletion/duplication, mosaicism is a possible cause.
Most MS-MLPA probes detect the methylation of the first Cytosine nucleotide in a single HhaI site within the
sequence detected by the probe (GmCGC). If methylation is absent for this particular CpG-site, it does not
necessarily mean that the whole CpG island is unmethylated! We have no data showing that methylation detected
by a particular probe indeed influences the mRNA level of that gene.
A point mutation within the HhaI site of a MS-MLPA probe may result in a false positive methylation signal as the
HhaI enzyme will not be able to digest the probe-sample DNA hybrid.
The great majority of CG sequences in the human genome located outside CpG islands is methylated. CG
sequences located within CpG islands are often protected against methylation, but the methylation status may
differ between tissues and between age groups. Sequences that are methylated in blood-derived DNA might be
unmethylated in e.g. amniotic fluid-derived DNA.
The optimal cut-off values for detecting a significant change in methylation of a sequence is probe dependent and
is dependent on sample type and application. In certain cases there might be a significant variation in methylation
of specific sequences between different normal individuals.
For many CpG islands located within imprinted chromosomal regions, one parental copy of the CpG island is
methylated while the copy inherited from the other parent is unmethylated. The average methylation quotient is
therefore 0.5. When testing samples for imprinted diseases, the threshold value for abnormal methylation can be
determined by testing sufficient DNA samples from healthy individuals.
MS-MLPA probes detecting sequences in CpG islands outside imprinting regions, e.g. in promoter regions of
genes, often reproducibly yield a low residual signal in the digested reference samples. This might be due to
methylation of the sequence in a small percentage of the cells tested. This background signal is often higher in
probes detecting sequences near the edge of the CpG island. Similarly, sequences located near the edge of a CpG
island more easily lose their protection against methylation and often show a higher frequency of methylation in
tumours.
Most MLPA probemixes contain reference probes. Copy number changes detected by reference probes are
unlikely to be related to the condition tested for. The identity of reference probes is available on request.
Analysis of parental samples might be necessary in certain cases for correct interpretation of results.
MLPA products are not CE/FDA certified. Internal MLPA technique validation in your laboratory is essential.

MS-MLPA results are more plausible when:

The overall standard variation per probe in the reference samples is low (<10%).
Probes show a decreased or increased signal for adjacent exons (multi-exon deletion or duplication).
The same result is obtained in a new MS-MLPA run using less DNA (if possible) or using different reference
samples. When less DNA is used, possible contaminants which may influence the probe signal are diluted.
An MS-MLPA result is unlikely to be reliable when:
Probes for non-neighbouring exons show a decreased or increased signal, e.g. deletion of exon 3 and 17.
In the same sample, one or more reference probes show an abnormal copy number.
Many abnormalities are found in a patient cohort of a disease in which copy number changes are known to be
rare.
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9. MS-MLPA PROTOCOL FOR DNA ANALYSIS IN A NUTSHELL

1. DNA DENATURATION
Heat a 5 l DNA sample for 5 minutes at 98o C
2. HYBRIDISATION OF PROBES TO SAMPLE DNA
Cool down to room temperature, open the tubes
Add a mixture of 1.5 l SALSA probemix and 1.5 l MLPA buffer and mix
Incubate 1 minute at 95o C + 16 hours hybridisation at 60o C
3. LIGATION AND LIGATION-DIGESTION OF HYBRIDISED PROBES
Reduce thermocycler temperature to 20o C, open tubes
Add 3 l Ligase buffer A and 10 l dH2O to each tube and mix.
Transfer 10 l to a second tube
Place samples in thermocycler, heat to 48o C
Add 10 l Ligase-65 master mix to the first tube (undigested reaction)
Add 10 l Ligase-Digestion master mix to the second tube (digested reaction)
Incubate 30 minutes at 48o C
Heat to inactivate the ligase enzyme: 5 minutes 98oC
4. PCR AMPLIFICATION OF LIGATED PROBES
Cool down to room temperature, open tubes
Transfer 10 l to a new tube
Add 4 l SALSA PCR buffer + 26 l dH2O to each tube and mix
Add 5 l polymerase mix at room temperature
Start PCR
5. CAPILLARY ELECTROPHORESIS OF PCR PRODUCTS
6. ANALYSE RESULTS
Determine RELATIVE size of the fluorescent peaks
Compare results to reference samples
Ligase-65 mix: 1.5 l Ligase buffer B + 8.25 l dH2O + 0.25 l Ligase-65
Ligase-Digestion mix: 1.5 l Ligase buffer B + 7.75 l dH2O + 0.25 l Ligase-65 + 0.5 l HhaI
Polymerase mix: 3.75 l dH2O + 1 l PCR primer mix + 0.25 l SALSA polymerase

PREVENT FALSE POSITIVE OR NEGATIVE RESULTS. READ THE COMPLETE PROTOCOL!

10. EXPLANATION OF SYMBOLS USED

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Manufacturer

Store at

Lot No.

Keep away from heat or

direct sunlight

Use by

Cat. No.

No. of Tests

Read instructions before

use

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