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MRC-Holland Description version 18; 26-11-2012 ® MLPA SALSA MLPA probemix P018-F1 SHOX Lot F1-0411: As compared to version E1 (lot E1-1009), three new probes near the PAR1 boundary have been included. One probe (GPR143) has been removed. The 88 and 96 nt control fragments have been replaced (QDX2). Léri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. Deletions of the SHOX gene and/or regulatory elements downstream of SHOX, are detected in approximately 60% of LWD patients (Benito-Sanz et al. 2006; Huber et al. 2006; Fukami et al. 2006; Chen et al. 2009). The SHOX gene and the regulatory elements downstream of SHOX, are located within the PAR1 region (pseudoautosomal region 1) which covers the 3000 kb of the X and Y chromosomes next to the p-telomere. This P018-F1 SHOX probemix contains probes for each exon of the human SHOX gene, as well as a probe just before the SHOX promoter region. In addition, several probes are present detecting sequences in a region downstream of SHOX which has been implicated in regulation of SHOX transcription. Furthermore, several probes on the X chromosome are included in this probemix that can be used to characterise larger deletions and to distinguish SHOX deletions from a Turner syndrome karyotype. Finally, ten autosomal reference probes are included. This SALSA® MLPA® probemix is designed to detect copy number changes of one or more sequences in the aforementioned gene and chromosomal regions in a DNA sample. For sequences within the PAR regions and the autosomal reference probes, heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. Deletions of a probe’s recognition sequence on the X-chromosome, outside the PAR regions, will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognisable by a 35-50% reduction in relative peak area. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak area, 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. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that many defects in the SHOX gene are expected to be small (point) mutations which will not be detected by this SALSA® MLPA® test. 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 P329 CRLF2-CSF2RA-IL3RA: contains probes for these PAR1 region genes. Deletions of the IL3RA and CSF2RA genes, resulting in CRLF2 overexpression, are found in Acute Lymphoblastic Leukaemia samples. P216 GHD: contains probes for GH1, PROP1, POU1F1, GHRHR, HESX1, LHX3 and LHX4. P217 IGF1R: contains probes for IGF1R and IGFBP3. P262 GHI: contains probes for IGF1, GHR, JAK2 and STAT5B. P026 Sotos: contains probes for NSD1. P384 Human Height: contains probes for HHIP, HMGA2, ADAMTSL3, NPPC, IHH. More information Website E-mail Mail : www.mlpa.com : [email protected] (information & technical questions); [email protected] (for orders) : MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA MLPA probemix P018 SHOX Page 1 of 8 MRC-Holland Description version 18; 26-11-2012 ® MLPA References for SALSA® MLPA® probemix P018 SHOX Chen, J. et al. (2009). Enhancer mutations of the SHOX gene as a frequent cause of short stature - the essential role of a 250 kb downstream regulatory domain. J.Med.Genet. on line Benito-Sanz S. et al. (2006). PAR1 deletions downstream of SHOX are the most frequent defect in a Spanish cohort of Leri-Weill dyschondrosteosis (LWD) probands. Hum Mutat. 2006 Oct;27(10):1062. Gatta V. et al. (2006). Identification and characterization of different SHOX gene deletions in patients with Leri-Weill dyschondrosteosys by MLPA assay. J Hum Genet. 2006 Nov 8. Fukami, M. et al. (2006). Transactivation function of an approximately 800-bp evolutionarily conserved sequence at the SHOX 3' region: implication for the downstream enhancer. Am. J. Hum. Genet. 78: 167-170. Huber et al. (2006). High incidence of SHOX anomalies in individuals with short stature. J Med Genet. 43(9):735-9. Schiller et al. (2000) Phenotypic variation and genetic heterogeneity in Léri-Weill syndrome. Eur. J. Hum. Genet. 8: 54-62 Exon numbering The exon numbering from the Genbank mRNA reference sequences is used in this product description. This exon numbering is different from the SHOX exon numbering in many articles. At least 2 alternatively spliced SHOX mRNA forms are known, encoding proteins with different patterns of expression. The 2 mRNAs, SHOXa and SHOXb, encode for proteins of 292 and 225 amino acids, respectively. Both transcripts have a common 5' end. Transcript variant 1, as exemplified by the Genbank NM_000451.3 reference sequence, contains exons 1-6. Transcript variant 2, as exemplified by reference sequence NM_006883.2, contains exons 1-5 + 7. Exon 6 is called 6a and exon 7 is called exon 6b in most SHOX articles. Exon 7 contains the last 42 nt of the variant 2 coding sequence. As this exon 7 sequence is entirely composed of two Alu repeats, we designed a probe in a unique intron sequence located in front of exon 7. Data analysis The P018-F1 SHOX probemix contains 45 MLPA probes with amplification products between 130 and 490 nt. In addition, it contains 10 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 (Dfragments) at 88-92-96 nt, one X-fragment at 100 nt and two Y-fragment at 105 nt and 118 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can be intra-normalized by dividing the peak area of each amplification product by the total area of only the reference probes in the probemix (block normalization). Ratios can be obtained by dividing the intra-normalized probe ratio in a sample by the average intra-normalized probe ratio of all reference runs. This type of normalization assumes that no changes occur in the genomic regions targeted by the reference probes. It is strongly recommended using reference and patient samples of the same sex to minimize variation, as intersex comparison makes analysis more difficult. Sex determination can also be done by visual examination of the electropherogram. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blots or long range PCR. Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge from our website www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://projects.tcag.ca/variation. For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed at MRC-Holland. Info/remarks/suggestions for improvement: [email protected]. SALSA MLPA probemix P018 SHOX Page 2 of 8 MRC-Holland ® MLPA Description version 18; 26-11-2012 Table 1. SALSA MLPA P018-F1 SHOX probemix Length (nt) 64-70-76-82 88-92-96 * 100 105 118 * SALSA MLPA probe Chromosomal position reference Outside PAR1 SHOX region / PAR1 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 (AMOT gene) Y-fragment: Specific for the Y chromosome (UTY gene) Y-fragment S0135-L16766: Specific for the Y chromosome (ZFY gene) 130 Reference probe 00797-L00463 5q31 136 SHOX-area probe 05642-L05096 Xp22.33-PAR1 142 IL3RA probe 13597-L15055 Xp22.33-PAR1 148 SHOX-area probe 05648-L06218 Xp22.33-PAR1 154 SHOX-area probe 13821-L14642 Xp22.33-PAR1 160 Reference probe 04966-L04696 1p22 166 SHOX probe 01145-L00702 Exon 1 PAR1 172 SHOX-area probe 05643-L15705 Xp22.33-PAR1 178 ¥ SHOX-area probe 05649-L20176 Xp22.33-PAR1 185 ¥ SHOX-area probe 06293-L20177 Xp22.33-PAR1 191 ¥ Reference probe 06057-L05512 4p16 199 ¥ SHOX-area probe 13296-L20175 Xp22.33-PAR1 204 SHOX probe 01146-L06220 Exon 2 PAR1 211 ~ PPP2R3B probe 09333-L10292 Xp22.33-PAR1 219 Reference probe 03247-L02684 13q14 226 ¥ SHOX probe 09336-L20178 Intron 6 PAR1 231 SHOX probe 09337-L00911 Exon 6 (6a) PAR1 238 + KAL1 probe 06402-L09795 Xp22.31 245 SHOX probe 01147-L00802 Exon 3 PAR1 254 **+ ARSF probe 16846-L20647 Xp22.33 261 ¥ Reference probe 00587-L20649 18q21 266 ¥ LOC159015 probe 01341-L20651 4 kb before SHOX-PAR1 274 + FANCB probe 03906-L03066 Xp22.2 283 + NLGN4X probe 05587-L04577 Xp22.31 290 SHOX-area probe 06291-L06222 Xp22.33-PAR1 300 SHOX probe 01148-L15501 Exon 4 PAR1 310 ~ ASMT probe 01153-L00712 Xp22.33-PAR1 318 SHOX-area probe 05645-L05099 Xp22.33-PAR1 328 **+ PRKX probe 16898-L19768 Xp22.33 337 ¥ SHOX probe 01149-L19676 Exon 5 PAR1 346 Reference probe 06560-L06118 1q32 VAMP7 (SYBL1) probe 01156-L00659 Xq28-PAR2 355 ∞ 369 ¥ Reference probe 01838-L19680 16p13 377 ¥ SHOX-area probe 14697-L19677 Xp22.33-PAR1 386 CSF2RA probe 10251-L15502 Xp22.33-PAR1 392 SHOX probe 09338-L15503 Intron 6 PAR1 403 ¥ CRLF2 probe 13911-L19678 Xp22.33-PAR1 412 Reference probe 09793-L12593 15q21 420 + AIFM1 (PDCD8) probe 00820-L15506 Xq26.1 432 SHOX-area probe 05646-L15507 Xp22.33-PAR1 442 ¥ SHOX-area probe 09335-L19679 Xp22.33-PAR1 453 ** ZBED1 probe 16858-L15742 Xp22.33-PAR1 463 SHOX-area probe 13297-L15510 Xp22.33-PAR1 474 Reference probe 09888-L10301 16p13 490 ± Reference probe 12463-L13464 9q31 * Control fragments 88, 96 and 118 have been replaced in version F1 (from lot F1-0411 onwards). ** New in version F1 (from lot F1-0411 onwards). ¥ Changed in version F1 (from lot F1-0411 onwards). Change in length but no change in sequence detected. + X-chromosome, outside PAR region. Gives 50 % reduced signal in males as compared to females. SALSA MLPA probemix P018 SHOX Page 3 of 8 MRC-Holland ® MLPA Description version 18; 26-11-2012 ~ This probe has been found to be duplicated in an apparently healthy individual in one of our quality tests. ∞ This probe has been found to be deleted in an apparently healthy individual in one of our quality tests. ± More variable. This probe has a high standard deviation Table 2. SHOX probes arranged according to chromosomal location Length SALSA MLPA (nt) probe Gene Exon Ligation site NM_000451.3 Partial sequence (24 nt adjacent to ligation site) Distance to next probe P-telomere / start PAR1 region PPP2R3B gene CGTCCGAGTTCC-ACTCGCGCTACA SHOX region 4.7 kb before start GCCTGGAACAGA-ACTTCCGCGGGG LOC159015 SHOX gene 227 kb 273.0 kb 211 ~ 09333-L10292 266 01341-L20651 166 204 245 300 337 231 01145-L00702 01146-L06220 01147-L00802 01148-L15501 01149-L19676 09337-L00911 SHOX stopcodon 1568-1570 (ex 6) 226 09336-L20178 SHOX intron 6 392 09338-L15503 SHOX intron 6 6.4 kb after exon 6 1.4 kb before exon 7 (only present in NM_006883.2) 136 154 172 199 318 § 432 § 463 § 290 § 185 § 148 178 442 377 403 386 142 310 ~ 453 05642-L05096 13821-L14642 05643-L15705 13296-L20175 05645-L05099 05646-L15507 13297-L15510 06291-L06222 06293-L20177 05648-L06218 05649-L20176 09335-L19679 14697-L19677 13911-L19678 10251-L15502 13597-L15055 01153-L00712 16858-L15742 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 Xp22.32-PAR1 CRLF2 gene CSF2RA gene IL3RA gene ASMT gene ZBED1 gene GCAGCAGTGAAA-GTGAGCATTCCC GATGGCTGATAA-TTACTCCGTATG ACACCACCAGAGT-TACTTGAATCAA GGAAAACCACGT-TCCTATCGATCC TGTTCCCACCGT-AAAACTCACTCC TGCATGTCTGCT-TTTTGAATGGCC TACAGCAAATGA-TACGTATAAATT CTTGAAAGGGCA-GGAACTCTAATT TAATTGATGAGA-TGCAGAAGCCAG TGGTGCTGAAAT-GAGGAAGCCCTG TGAGGAGGTACC-TCAAAGCTAAAC GAAATTCAGTTT-TAATAACACAGA CTCTGGTGAGAT-GCCATCTAGAGA GAATGCCAGCAA-ATACTCCAGGAC GACAAGCCTTCT-GCTCTGTGAGTT TGCACAGATAAG-TTTGTCGTCTTT GACATCCCAGAA-GTGGTGTGGACG TCGTCAAGAGCA-ACACGGAGCAGA 19.8 11.4 45.4 57.7 8.5 10.7 6.3 0.4 15.4 48.7 64.4 66.0 338.0 73.9 69.8 280.7 706.4 593.8 kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb kb 254 + 328 + 283 + 238 + 274 + 420 + 16846-L20647 16898-L19768 05587-L04577 06402-L09795 03906-L03066 00820-L15506 ARSF gene PRKX gene NLGN4X gene KAL1 gene FANCB gene AIFM1 gene End of PAR1 region Xp22.33 CATCCATATAAT-TATGGGTTTGAC Xp22.33 CGATTAGGAAAC-ATGAAGGTCAGT Xp22.31 GACGGCTTGGGT-GATGCACGAAAT Xp22.31 GTTTCCTGAAGC-GTGTGCCCACAA Xp22.2 TCTCATCAGAAT-TCTCCCTATAAA Xq26.1 TATTGGTCTTGT-GGACAGTAGTTT Start of PAR2 region 536.9 2606.5 2311.9 6304.2 114321.4 25732.3 kb kb kb kb kb kb 355∞‡ 01156-L00659 VAMP7 gene (PAR2) SHOX startcodon 692-694 (ex 2) SHOX exon 1 SHOX exon 2 SHOX exon 3 SHOX exon 4 SHOX exon 5 SHOX exon 6 (6a) 99-100 920-921 1032-1034 1198-1199 1260-1261 1506-1507 Xq28 4.7 kb TTTCTACTGCAA-ACAGAAATGGGA ACCACGTAGACA-ATGACAAGGAGA CGGGCAGACCAA-GCTGAAACAGAG CAGAACCGGAGA-GCCAAGTGCCGC ACAGCCAACCAC-CTAGACGCCTGC AAGCAACAGCAA-GAATTCCAGCAT 6.7 3.6 6.2 0.2 3.5 6.4 TGGCTTCACGAG-TTCAGCCCATTG 6.4 kb TCCCACATTCTT-GGAATCACAATG 56.8 kb TGTGGGAAAAGT-GTTTCCATTCTG Very close to q-telomere + X-chromosome, outside PAR region. Gives halve the signal in males as compared to females. ~ This probe has been found to be duplicated in an apparently healthy individual in one of our quality tests. ∞ This probe has been found to be deleted in an apparently healthy individual in one of our quality tests. ‡ The VAMP7 probe at 355 nt is located very close to the q-telomere of X and Y in the PAR2 pseudoautosomal region. § These five probes are in the putative SHOX regulatory region (CNE7-9) described by Chen J. (2009) J.Med.Genet. online. The 290 and 185 nt probes are within the putative regulatory region identified by Fukami, M. et al (2006) Am. J. Hum. Genet. 78: 167-170. SALSA MLPA probemix P018 SHOX kb kb kb kb kb kb Page 4 of 8 MRC-Holland Description version 18; 26-11-2012 ® MLPA Note: Exon numbering is according to the NCBI NM_ reference sequences and might be different from the literature! Several probes are only included to facilitate determination of the extent of a deletion / duplication. Copy number alterations of certain probes may not be related to the condition being tested. Complete probe sequences are available on request: [email protected]. Please notify us of any mistakes: [email protected]. The following changes in the relative probe signals (as compared to the autosomal reference probes indicated in table 1) are expected when MALE reference DNA is used: 1. An approx. 100% increase in signal of the chromosome X probes outside the PAR regions (marked + in table 2) and a complete loss of the Y probe signals in female DNA. The signals of the SHOX probes, the other probes in the PAR1 region and the VAMP7 (SYBL) probe in the PAR2 region at the q telomere should remain unchanged as the Y chromosome also contains a copy of the PAR1 and PAR2 regions. 2. A 40-60% reduction in signal of one or more SHOX probes, in case of a (partial) deletion of the SHOX gene. These deletions will in general cause Leri-Weill dyschondrosteosis. 3. A 40-60% reduction in signal of one or more probes downstream of SHOX, indicated with Xp22.32-PAR1 in table 2. Deletions in this region have been associated with Leri-Weill dyschondrosteosis (Benito-Sanz et al (2005) Am. J. Hum. Genet. 77, 533-544). We included numerous probes in this region, as the exact location of the SHOX regulatory region is not 100% certain. The most likely region is marked with § in table 2. Please note that not all deletions detected by the Xp22.32-PAR1 probes will result in Leri—Weill dyschondrosteosis. 4. A 40-60% reduction in signal of the SHOX probes and all other PAR1 probes and a complete loss of one or more other Xp probes in case of deletions of Xp that extend outside PAR1. 5. A complete loss of the Y probe signals and a 40-60% reduction of the SHOX, the PAR1 and the PAR2 probes in Turner syndrome (X0). 6. An approx. 100 % increase in signal of the X probes outside the PAR regions and a 40-60% increase in signal of the SHOX and the PAR1 and PAR2 probes in Klinefelter syndrome (XXY). 7. An approx. 200 % increase in signal of the X probes outside the PAR regions, the disappearance of the Y probe signals and a 40-60% increase in signal of the SHOX and the PAR1 and PAR2 probes in Triple X syndrome (XXX). The following changes in the relative probe signals (as compared to the autosomal reference probes indicated in table 1) are expected when FEMALE reference DNA is used: 1. A 40-60% decrease in signal of the chromosome X probes outside the PAR regions (marked + in table 2) and the appearance of Y probe signals in male DNA. The signals of the SHOX probes, the other probes in the PAR1 region and the VAMP7 (SYBL) probe in the PAR2 region at the q telomere should remain unchanged as the Y chromosome also contains a copy of the PAR1 and PAR2 regions. 2. A 40-60% reduction in signal of one or more SHOX probes, in case of a (partial) deletion of the SHOX gene. These deletions will in general cause Leri-Weill dyschondrosteosis. 3. A 40-60% reduction in signal of one or more probes downstream of SHOX, indicated with Xp22.32-PAR1 in table 2. Deletions in this region have been associated with Leri-Weill dyschondrosteosis (Benito-Sanz et al (2005) Am. J. Hum. Genet. 77, 533-544). We included numerous probes in this region, as the exact location of the SHOX regulatory region is not 100% certain. The most likely region is indicated in table 2. Please note that not all deletions detected by the Xp22.32-PAR1 probes will result in Leri—Weill dyschondrosteosis. 4. A 40-60% reduction in signal of the SHOX probes and all other PAR1 probes and a 40-60% decrease in signal of one or more other Xp probes in case of deletions of Xp that extend outside the PAR1 region. 5. A 40-60% reduction of all chromosome X probes in Turner syndrome (X0). 6. The appearance of Y probe signals and a 40-60% increase in signal of the SHOX and the PAR1 and PAR2 probes in Klinefelter syndrome (XXY). 7. A 40-60 % increase in signal of all chromosome X probes (all probes in table 2) in Triple X syndrome (XXX). SALSA MLPA probemix P018 SHOX Page 5 of 8 MRC-Holland ® MLPA Description version 18; 26-11-2012 SALSA MLPA probemix P018-F1 SHOX sample pictures 40000 96.44 86.23 35000 266.26 230.57 91.16 105.55 30000 244.81 184.27 25000 217.99 198.22 177.37 171.49 190.37 134.01 128.19 158.06 151.73 164.55 146.12 141.10 20000 115.01 260.31 451.81 385.75 289.41 376.64 392.97 309.24 224.73 354.10 345.16 211.18 203.81 433.28 490.99 336.56 253.67 15000 474.87 441.95 326.25 237.65 420.39 281.43 274.12 10000 D ye S ign al 463.82 403.13 316.99 100.71 412.00 368.40 298.91 5000 0 50 100 150 200 250 300 350 400 450 500 Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P018-F1 SHOX (lot F1-0411). The old MLPA buffer (replaced in December 2012) was used. Vials with the old MLPA buffer have a white label. 50000 184.29 198.20 45000 217.99 40000 128.27 35000 158.10 171.53 190.40 151.79 177.40 134.11146.20 86.31 96.51 260.26 224.72 211.18 141.18 230.60 30000 91.20 105.60 266.23 25000 244.82 289.38 20000 309.21 100.75 115.08 164.57 298.89 203.82 15000 D ye S ign al 316.96 253.62 393.04 385.79 376.71 412.08 368.48 403.18 451.79 433.26 463.78 336.61 326.27 237.63 10000 354.13 345.16 420.39 281.40 274.10 441.99 490.92 474.80 5000 0 50 100 150 200 250 300 350 400 450 500 Size (nt) Figure 2. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P018-F1 SHOX (lot F1-0411). The new MLPA buffer (introduced in December 2012) was used. Vials with the new MLPA buffer have a yellow label. SALSA MLPA probemix P018 SHOX Page 6 of 8 MRC-Holland ® MLPA Description version 18; 26-11-2012 85.97 35000 96.33 266.23 230.43 30000 253.65 100.61 184.25 91.07 244.66 25000 224.69 198.25 217.92 211.15 237.64 260.31 177.35 158.08 171.50 133.97 151.73 128.17 190.38 146.11 164.54 203.80 141.11 20000 309.19 289.34 326.21 451.88 420.46 385.87 376.67 354.13 368.39 345.20 281.35 298.81 274.07 316.98 393.00 412.00 433.43 463.84 403.19 491.03 336.47 474.92 15000 441.87 D ye S ign al 10000 5000 0 50 100 150 200 250 300 350 400 450 500 Size (nt) Figure 3. Capillary electrophoresis pattern from a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P018-F1 SHOX (lot F1-0411). The old MLPA buffer (replaced in December 2012) was used. Vials with the old MLPA buffer have a white label. 45000 184.27 198.26 40000 260.26 217.93 158.09 134.00 151.75 171.52 128.20 35000 211.17 100.66 96.36 25000 224.68 177.36 146.13 141.15 86.04 30000 190.38 230.45 266.19 91.11 253.60 244.66 20000 289.30 309.19 326.21 237.62 281.32 298.82 164.58 15000 203.82 274.03 316.97 393.03 385.87 420.46 376.70 354.14 412.02 368.43 433.38 345.19 403.18 451.85 463.82 491.04 336.50 441.88 474.89 D ye S ign al 10000 5000 0 50 100 150 200 250 300 350 400 450 500 Size (nt) Figure 4. Capillary electrophoresis pattern from a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P018-F1 SHOX (lot F1-0411). The new MLPA buffer (introduced in December 2012) was used. Vials with the new MLPA buffer have a yellow label. SALSA MLPA probemix P018 SHOX Page 7 of 8 MRC-Holland Description version 18; 26-11-2012 ® MLPA Implemented Changes –compared to the previous product description version(s). Version 18 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 17 (48) - Various minor textual changes. - Mistake corrected in table 2, 337 nt probe 01149-L19676 (1261-1262 changed into 1260-1261). - Data analysis method has been modified. Version 16 (46) - Additional related SALSA MLPA probemixes added on page 1. - Minor textual changes on page 7 (Implemented Changes...). Version 15 (45) - This product description has been changed to incorporate a new version (lot number added, changes in Table 1 and Table 2, new pictures included). Version 14 (45) - Minor textual change of page 5 (The following changes….). Version 13 (45) - This product description has been changed to incorporate a new version (lot number added, changes in Table 1 and Table 2, new pictures included). - Various minor textual changes on page 1; various minor layout changes. Tables have been numbered. - Small changes of probe lengths in Table 1 & 2. SALSA MLPA probemix P018 SHOX Page 8 of 8