Download P003 MLH1 / MSH2 EXON DELETION TEST KIT:

MRC-Holland
Vs. 04; 22-11-2007
®
MLPA
SALSA MLPA kit PM200 Mouse control
Lot 0507; 0806
Mouse models can be generated using different methods, conventionally by gene targeting or by
oligonucleotide targeting of ES cells. Both of these methods rely on the homologous integration of a stretch
of foreign DNA (construct or oligonucleotide) to modify the genome. In general, the construct is delivered to
the ES cells by electroporation and eventually integrates in the genome through homologous recombination,
although non-homologous integration is observed more frequently.
MLPA can be used not only for the detection of homologous integration events in conventionally targeted ES
cell clones, but also provides a powerful tool in bacterial artificial chromosome (BAC) recombineering,
transgenesis and targeting. In addition, once available, a kit can be used for high throughput genotyping of
different mice strains. Major advantages of MLPA over Southern blotting and PCR for the screening of
targeted ES cells are (i) changes as small as a single nucleotide exchange can be studied using a minimum
of 20 ng sample DNA, (ii) owing to its simple procedure, large numbers of samples can be analyzed
simultaneously with a minimum of hands-on work and (iii) MLPA is quantitative and can discriminate
between homologous, non-homologous and incomplete recombination events. Multiple integrations can be
identified as well.
This probe mix contains 6 MLPA control probes, which should be supplemented with synthetic probes
specific for the sequence of interest. Synthetic probes can be designed using our synthetic probe design
protocol , which can be downloaded for free from our website. A list of the name, length and target specific
sequence of the SALSA MLPA PM200 control probes can be found on page 3. Please carefully read the
description of how to prepare and add the synthetic target-specific MLPA probes, and how to perform the
MLPA reaction.
MLPA® kits are sold by MRC-Holland for research purposes and to demonstrate the possibilities
of the MLPA technique. This kit is not CE/FDA certified for use in diagnostic procedures. SALSA
MLPA kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA MLPA
test kits includes a limited license to use these products for research purposes.
The use of this MLPA kit requires a thermocycler with heated lid and sequence type electrophoresis
equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in
Nucleic Acid Research 30, e57 (2002). The MLPA mouse application has been first described by Langerak et
al. Rapid and quantitative detection of homologous and non-homologous recombination events using three
oligonucleotide MLPA. Nucleic Acids Res., Dec 2005; 33: e188.
More information
Website
E-mail
Fax
Mail
:http://www.mlpa.com
:[email protected]
:+31-20-6891149
:MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands
SALSA MLPA kit PM200 Mouse control
Page 1 of 4
MRC-Holland
Vs. 04; 22-11-2007
®
MLPA
Data analysis
The PM200 Mouse control probemix contains 6 different probes with amplification products between 175 and
545 nt, as well as 4 control fragments generating an amplification product smaller than 90 nt. In many
cases, resul;ts can be confirmed. Southern blot, FISH or long range PCR. The length of the amplification
products does not include the 3’ Adenine, which is usually added by a non-proofreading polymerase such as
the SALSA polymerase.
MRC-Holland usually defines relative probe signals by dividing each measured peak area by the sum of all
peak areas of that sample. The ratio of each peak’s relative probe area is then compared to that obtained on
a DNA control sample. Note that in case of large deletions, this method of analysis will result in a less than
50% reduction of relative probe signal. An alternative method is to define relative probe signals of probes by
comparing them to the combined signals of the control probes. When only small numbers of samples are
tested, visual comparison of peak profiles should be sufficient to easily identify exon deletions. Comparison
of results should preferably be performed within one experiment, since intra-assay variation is larger. Only
samples purified by the same method should be compared.
The column with the Gene name shows the HUGO name of the gene detected by the probe. In many cases
genes are better known by other alias names. The HUGO name and the alias names of each gene can be
found on http://www.ncbi.nlm.nih.gov/Omim/searchomim.html.
Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge
from our website www.mlpa.com.
Control fragments
Each SALSA MLPA probemix includes four DQ (DNA Quantity) control fragments. The amplification products
generated by these DQ fragments are 64, 70, 76 and 82 nt long and thus much shorter than the
amplification products generated by the probes. The purpose of these short DQ fragments is to give a
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 ligation did not take place. 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 nt DQ
amplification products will be hardly visible.
When 50 ng human DNA is used in the MLPA reaction, the DQ fragments will be visible, but with a much
smaller peak size than the other amplification products. However, in case the 64-70-76-82 nt DQ
amplification products have peak sizes which are larger than or comparable to those of the other probes,
either the ligation reaction has failed or the amount of sample DNA was less than 20 ng. In either case, the
results obtained may not be reliable.
This probemix was developed by A.O.H. Nygren at MRC-Holland. In case the results obtained with this
probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could
be made a coauthor.
Info/remarks/suggestions for improvement: [email protected].
SALSA MLPA kit PM200 Mouse control
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MRC-Holland
Vs. 04; 22-11-2007
®
MLPA
Notes for optimal capillary electrophoresis results
This probe mix contains only 6 MLPA probes; even when supplemented with one to four synthetic MLPA
probes, the peak signal could be so high that the signals could saturate the detector of the instrument used.
In order to generate reliable and quantitative results, the injection time can be reduced with as much as two
thirds of the injection time.
Probe mix preparation
Dilute each synthetic oligonucleotide separately to final concentration of 1 μM. Mix 0.8 μl of each 1 μM
dilution in a total volume of 300 μl (Supplement mix).
MLPA reaction
How to perform an MLPA reaction is described in the protocol: DNA- Detection/Quantification Protocol that
can be obtained at www.mlpa.com unless supplemented with the reagents. In this application there are a
few deviations from the standard protocol and these are explained below
1) DNA samples: As the DNA concentrations are usually quite high we recommend not using more than
1μl DNA in a total volume of 5μl TE-Buffer for each MLPA reaction. If the DNA concentration is known we
recommend using 50 ng in 5 μl volume.
2) Probe mix preparation: For every MLPA reaction prepare 1.0 μl SALSA PM200 and 0.5 μl of the
Supplement mix.
SALSA MLPA kit PM200 Mouse control
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MRC-Holland
®
MLPA
Vs. 04; 22-11-2007
SALSA MLPA PM200 Mouse control probemix
Length (nt)
SALSA MLPA probe
HUGO name Chromosomal
position
Genbank
64-70-76-82*
DQ-control fragments
175
probe 3459-L3019
IFNG
10 D2
NM_008337
229
probe 3474-L3026
TLR2
3 E3
NM_011905
295
probe 3477-L3033
F2R
13 D1
NM_010169
337
probe 3451-L3037
SELE
1 H2.2
NM_011345
409
probe 3483-L3044
TBP
17 A2
NM_013684
454
probe 3454-L3050
HP
8 D3
NM_017370
* Not ligation-dependent; these fragments indicate the amount of DNA used.
Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes. The
identity of the genes detected by the control probes is available on request: [email protected].
The Genbank sequences can be found on the Genbank website:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide.
SALSA MLPA kit PM200 Mouse control Sample picture
Lot 0806
Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng mouse tail DNA analyzed
with SALSA MLPA kit PM200 Mouse control (lot 0806). The open space between ~88 and 132 nt (see our
synthetic probe design protocol) is the region reserved for self-made synthetic MLPA probes, which can be
added to the probemix.
SALSA MLPA kit PM200 Mouse control
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