MRC-Holland
Description version 07; 03 June 2015
®
MLPA
SALSA MLPA probemix P356-A2 Chromosome 22q
Lot A2-0315. As compared to version A1 (lot A1-1009), three reference probes have been replaced. In
addition, the lengths of several probes and the control fragments (QDX2) have been adjusted.
This SALSA MLPA probemix can also be bought as part of the Telomere Follow-up Set. The Telomere Followup Set contains 12 Telomere follow-up probemixes: 465 telomere probes divided over 46 regions. The
introduction price (also valid in 2014-2015) for 12 × 25 reactions is € 1200.
This P356-A2 probemix can be used to confirm and further characterise abnormalities detected by SALSA®
MLPA® probemix P036 and/or SALSA® MLPA® probemixes P069 / P070 used for broad human subtelomere
screening.
This SALSA® MLPA® P356-A2 Chromosome 22q probemix contains:
19 probes in the DiGeorge syndrome region of chromosome arm 22q11
17 probes in the terminal 4.5 Mb of chromosome arm 22q
SALSA® MLPA® probemixes P036 and P069 / P070 each contain one MLPA probe for each subtelomeric
region. Most of these probes detect a well-characterised gene close to the telomere. The probes in P036 and
P069 / P070 are different, allowing results obtained with one probemix to be confirmed by the other. The
majority of probes in P069 and P070 are identical. For the p telomeres of the acrocentric chromosomes 13,
14, 15, 21 and 22, it is not possible to design reliable probes. For these chromosomes, the P069 probemix
only contains a probe for the q telomeric region. The P036 and P070 probemixes contain a second probe for
these q arms, which target a DNA sequence close to the centromere.
We recommend using SALSA® MLPA® probemix P036 Subtelomeres mix 1 and SALSA® MLPA® probemix
P069 Subtelomeres mix 2A / P070 Subtelomeres mix 2B for primary screening of the subtelomeres. Results
obtained with these probemixes can be confirmed and further characterized with one of the specific followup probemixes for telomere analysis, such as this P356-A2 Chromosome 22q probemix.
This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences in the
above mentioned chromosomal regions in a DNA sample. Heterozygous deletions of recognition sequences
should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a
mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak
height, even when not located exactly on the ligation site! In addition, some probe signals are more
sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and
duplications detected by MLPA should always be confirmed by other methods. Not all deletions and
duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature
when interpreting their findings.
SALSA® MLPA® probemixes and reagents are sold by MRC-Holland for research purposes and to
demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in
diagnostic procedures. Purchase of the SALSA® MLPA® test probemixes and reagents includes a
limited license to use these products for research purposes.
The use of a SALSA® MLPA® probemix and reagents 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).
Related SALSA® MLPA® probemixes
P250 DiGeorge: Contains a large number of probes for the 22q11 DiGeorge region
P324 22q11 mix 2: Contains additional 22q11 probes that are not present in the P250 DiGeorge probemix
P339 SHANK3: Contains probes for most SHANK3 exons
More information
Website : www.mlpa.com
E-mail
: info@mlpa.com (information & technical questions); order@mlpa.com (for orders)
Mail
: MRC-Holland bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands
SALSA MLPA probemix P356 Chromosome 22q
Page 1 of 5
MRC-Holland
®
MLPA
Description version 07; 03 June 2015
Related SALSA® MLPA® probemixes for specific subtelomeric analysis
P208
P230
P249
P264
P286
P291
P320
P358
P365
Human
Human
Human
Human
Human
Human
Human
Human
Human
Telomere-6: 2p, 3p, 6p, 8p
Telomere-7: 9p, 10p, 11p, 12p
Telomere-8: 17p, 18p, 19p, 20p
Telomere-9: 1q, 2q, 3q, 4q
Telomere-11: 9q, 10q, 11q, 12q
Telomere-12: 13q, 14q, 15q, 16q
Telomere-13: 17q, 18q, 19q, 20q
Telomere-15: 4p, 5p, 13q-cen, 14q-cen
Telomere-14: 7p,15q-cen, 16p, 21q-cen, 21q
P096 MR-2: 4p, 5p
P140 HBA: 16p
P147 1p36 deletions: 1p
P163 GJB: 13q-cen
P188 22q13: 22q
P250 DiGeorge: 22q11
P277 Human Telomere-10: 5q, 6q, 7q, 8q
P340 EHMT1: 9q
ME028 PWS/AS: 15q11
Data analysis
The P356-A2-0315 Chromosome 22q probemix contains 46 MLPA probes with amplification products
between 132 and 493 nt. In addition, it contains 9 control fragments generating an amplification product
smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA Denaturation
control fragments (D-fragments) at 88-92-96 nt, one X-fragment at 100 nt and one Y-fragment at 105. More
information on how to interpret observations on these control fragments can be found in the MLPA protocol.
Data generated by this probemix can first be normalised intra-sample by dividing the peak height of each
probe’s amplification product by the total peak height of only the reference probes in this probemix (block
normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe
ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this
type of normalisation assumes no changes occurred in the genomic regions recognised by the reference
probes.
Data normalisation should be performed within one experiment. Only samples purified by the same method
should be compared. Confirmation of most deletions and amplifications can be done by e.g. Southern
blotting, long range PCR, qPCR, FISH.
Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge
from our website www.mlpa.com.
Subtelomeric abnormalities
The detection of abnormal copy numbers in subtelomeric regions is very complicated. Compared to other
regions of the genome, these regions are rich in sequences with variable copy numbers. Copy number
changes of sequences within these regions can also occur in unaffected individuals and the effect of a
deletion or duplication will depend on the genes present in the affected region. The choice of genes targeted
by this probemix has in part been guided by their chromosomal location. Not all copy number changes
identified will result in phenotypic abnormalities!
For all abnormalities detected, we strongly recommend testing the patient’s parents to determine whether a
copy number aberration found in the patient is truly de novo. Please be aware that a considerable number of
abnormalities detected by a single probe may not have a phenotypic effect, but can be due to a rare
polymorphism or a copy number change which is also present in one of the parents. For some chromosome
arms, even a large subtelomeric deletion of more than 1 Mb can be inherited without a phenotypic effect.
No method will be capable of detecting all chromosomal aberrations. MLPA will not detect inversions or
balanced translocations. Frequent microdeletion syndromes can be detected with SALSA® MLPA® probemix
P245 Microdeletion Syndromes but many rare interstitial deletions will not be detected.
More information on copy number changes in the human genome can be found in the Database of Genomic
Variants (http://dgv.tcag.ca/dgv/app/home).
This probemix was developed by MRC-Holland.
Info/remarks/suggestions for improvement: info@mlpa.com.
SALSA MLPA probemix P356 Chromosome 22q
Page 2 of 5
MRC-Holland
®
MLPA
Description version 07; 03 June 2015
Table 1. SALSA MLPA P356-A2 Chromosome 22q probemix
Length
(nt)
64-70-76-82
88-92-96
100
105
132
139
148 «
154 «
160
165 «
178
184
190
196
202
208
214
220
228
234 «
242 *
247
252 «
260 «
266
274 «
280
287
292 «
301 «
310 «
319 «
328
337
346
353
362
373
382 «
391
400 «
409
418
428 «
448 «
454 *
466
474 «
484 «
493 *
SALSA MLPA probe
reference
Chromosomal position
22q11
22q13
Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA
D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation
X-fragment: Specific for the X chromosome
Y-fragment: Specific for the Y chromosome
Reference probe 00797-L21698
5q31
SLC25A18 probe 05457-L28211
22q11.21
SHANK3 probe 06786-L28212
PRODH probe 07485-L28213
22q11.21
HIRA probe 01214-L02328
22q11.21
GSC2 probe 05461-L16422
22q11.21
Reference probe 08599-L28166
17p11
MICAL3 probe 05458-L28169
22q11.21
Reference probe 10973-L28170
14q31
CLTCL1 probe 05462-L28171
22q11.21
CLDN5 probe 01218-L28172
22q11.21
BCL2L13 probe 03193-L28173
22q11.21
ALG12 probe 06733-L28502
GTSE1 probe 06080-L05535
22q13.31
USP18 probe 07528-L28175
22q11.21
SHANK3 probe 06787-L28176
Reference probe 08070-L28240
9p13
GAB4 probe 13888-L28372
22q11.1
MLC1 probe 06082-L28179
PLXNB2 probe 06083-L28239
CECR1 probe 05777-L28180
22q11.1
SBF1 probe 06084-L28181
Reference probe 09495-L28182
11q24
PEX26 probe 13954-L28183
22q11.21
MAPK8IP2 probe 05111-L28184
ARSA probe 02707-L28185
SHANK3 probe 20567-L14007
ACR probe 06087-L05542
RABL2B probe 06088-L05543
IL17RA probe 01082-L00660
22q11.1
Reference probe 11018-L11687
15q14
BID probe 13955-L15524
22q11.21
BID probe 01767-L16426
22q11.21
Reference probe 07816-L07546
3p22
ACR probe 09363-L05557
CECR2 probe 13953-L15492
22q11.21
SHANK3 probe 06785-L06377
Reference probe 04683-L04061
1p36
GTSE1 probe 06097-L06370
PRODH probe 07486-L16427
22q11.21
CECR5 probe 06251-L05757
22q11.1
Reference probe 02144-L01619
13q13
CDC45 probe 05463-L05808
22q11.21
CPT1B probe 06101-L16428
ACR probe 05112-L16429
22q13.33
Reference probe 14909-L27536
18p11
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.33
22q13.31
22q13.33
22q13.33
* New in version A2 (from lot A2-0315 onwards).
« This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to
incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA.
Note: In version A2 many probes have a small change in length, but no change in the sequence detected.
SALSA MLPA probemix P356 Chromosome 22q
Page 3 of 5
MRC-Holland
®
MLPA
Description version 07; 03 June 2015
Table 2. P356 probes arranged according to chromosomal location
Length
(nt)
SALSA MLPA
probe
Gene
247
337
448
266
391
139
208
353
362
184
287
228
428
154
165
196
160
466
202
13888-L28372
01082-L00660
06251-L05757
05777-L28180
13953-L15492
05457-L28211
03193-L28173
13955-L15524
01767-L16426
05458-L28169
13954-L28183
07528-L28175
07486-L16427
07485-L28213
05461-L16422
05462-L28171
01214-L02328
05463-L05808
01218-L28172
GAB4
IL17RA
CECR5
CECR1
CECR2
SLC25A18
BCL2L13
BID
BID
MICAL3
PEX26
USP18
PRODH
PRODH
GSC2
CLTCL1
HIRA
CDC45
CLDN5
Partial sequence (24 nt
adjacent to ligation site)
22q11 region
TAAAGCTCTGTA-ATATCAGGGACC
GCAGAGTTATCT-GTCCTGCAGCTG
CTCTGAAAGCCT-TCCGAAGGCTGG
CTGGTGAGGAAT-GTCACCTACAGG
TGACGTGGAGTT-TATCAGTGACCT
GCAGTGAGAAGA-GTCGAGTGAAGC
CACACTTCTCCA-GTGTTCAGCCCT
CATCCGGAATAT-TGCCAGGCACCT
CTACTGGTGTTT-GGCTTCCTCCAA
GAACTACCGCCT-GTCCCTGAGGCA
GAGCCTGGAGCT-GTGCTGGATGTG
CTCAGTCCCGAC-GTGGAACTCAGC
ACTACAGGGCCT-TCGGTGTCAGCG
CAGAGGCTTTGA-GAAGCCAGTGGG
GCTTTTCGTGCA-GAACCAGTATCC
TGTTGCCTTGGT-GACCGAGACCGC
GGAGCTGCTGAA-GGAGCTGCTACC
ATGTTCGTGTCC-GATTTCCGCAAA
TTCGCCAACATT-GTCGTCCGCGAG
220
418
214
252
260
274
474
292
301
310
400
148
234
382
484
319
328
06080-L05535
06097-L06370
06733-L28502
06082-L28179
06083-L28239
06084-L28181
06101-L16428
05111-L28184
02707-L28185
20567-L14007
06785-L06377
06786-L28212
06787-L28176
09363-L05557
05112-L16429
06087-L05542
06088-L05543
GTSE1
GTSE1
ALG12
MLC1
PLXNB2
SBF1
CPT1B
MAPK8IP2
ARSA
SHANK3
SHANK3
SHANK3
SHANK3
ACR
ACR
ACR
RABL2B
22q13 region
CTCTCCATGGAA-GGAGGCGGCGGC
CAGAATGGGACC-CGCCATGCTGCG
TCCAGCACAATT-ATGACAATTCAG
CTGCCAGGGGGT-TTGAGGACCCTC
CGAGGCTGCGGA-GACGCCTTTCAC
ATGCCACAGAGA-GGGAGGAAGAGG
GACGTAGGCTCT-GAGATAGCTGTC
CTGCAAACCTTA-TCCTCTATTCTT
GGAGGATCAGAT-CTCCGCTCGAGA
AAGCGGCGAGTT-TATGCCCAGAAC
GAGGAGAAAGAT-CGTGACCGGGAT
GGTCGGACACAA-GCAGGTGGTGGC
ACCAACTGTGAT-CAGTGAGCTCAG
ACCAACTGCCAT-TCTGCTGGTCTT
AGCTTGCTGAAT-TCACGATGGGTG
CAGAGGGAAATG-ACATTGCCCTCG
CTCCCACATTCT-AGGCCCGTGATA
Distance to ptelomere (hg18)
Distance to
next probe
15.824
15.960
16.011
16.068
16.337
16.423
16.552
16.602
16.607
16.705
16.946
17.013
17.299
17.304
17.517
17.622
17.699
17.847
17.891
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
136.2
50.8
57.6
268.6
86.7
128.5
50.4
4.5
98.0
241.6
66.7
285.6
5.4
213.2
104.4
77.4
148.5
43.8
27180.6
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
45.072
45.091
48.683
48.841
49.064
49.253
49.354
49.396
49.413
49.462
49.480
49.490
49.508
49.524
49.525
49.525
49.554
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
18.7
3592.7
157.1
223.4
189.0
101.3
42.2
16.9
48.6
18.3
10.0
17.3
15.9
1.2
0.4
28.8
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
kb
Note: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any
mistakes: info@mlpa.com.
SALSA MLPA probemix P356 Chromosome 22q
Page 4 of 5
MRC-Holland
Description version 07; 03 June 2015
®
MLPA
SALSA MLPA probemix P356-A2 Chromosome 22q sample picture
Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA
analysed with SALSA® MLPA® probemix P356-A2 Chromosome 22q (lot A2-0315).
Implemented Changes – compared to the previous product description versions.
Version 07 - 03 June 2015 (7)
- Product description adapted to a new product version (version number changed, lot number added,
small changes in Table 1 and Table 2, new picture included).
- Names of P036, P069 and P070 adjusted according to their name changes.
Version 06 (48)
- Warning added in Table 1, 256 nt probe 06083-L05538, 290 nt probe 05111-L04495, 297 nt probe
02707-L14005, 319 nt probe 06087-L05542 and 400 nt probe 06785-L06377.
Version 05 (48)
- Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added.
Version 04 (03)
- Various minor textual and layout changes.
- Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the
amplification products.
Version 03 (01)
- Information about the Telomere Follow-up Set has been added on page 1.
Version 02 (01)
- Various textual changes throughout the document.
- The section Related SALSA® MLPA® probemixes and Related SALSA® MLPA® probemixes for specific
subtelomeric analysis has been updated.
- Additional text added on p.2
Version 01 (45)
- Not applicable, new document.
SALSA MLPA probemix P356 Chromosome 22q
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