SALSA MLPA kit A071 - A078 DMD Lot 0606

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1 kit A071 - A078 DMD Lot 0606 Please note: This SALSA kit has been developed for the low-resolution detection of DMD (partial) deletions with an agarose gel electrophoresis or Agilent microfluidics. kit P034/P035 DMD is available for DMD screening using a capillary sequencer. DUCHENNE MUSCULAR DYSTROPHY and BECKER MUSCULAR DYSTROPHY can be caused by deletions, duplications or point mutations in the DMD gene on chromosome Xp21.2. The protein encoded by this gene is dystrophin. Information on DMD and the various deletions/duplications in this gene can be found on To what extent DMD manifests depends on whether the translational reading frame is lost or maintained. Partial gene deletions or duplications in the DMD gene account for as much as ~65% of cases of these dystrophies. This extremely high percentage may be due to the nature of the protein and the gene s extreme length (> 2.2 Mb). The A071-A078 DMD mixes contain one MLPA for each of the 79 DMD exons. In addition, a is present for the alternative exon 1: DP427c. These 80 s have been divided into eight different mixes from A071 to A078. In addition, in some mixes one or two reference s for other X chromosome sequences are present. Low-resolution detection of deletions in the DMD gene requires 8 different MLPA reactions for each DNA sample, whereas the use of the P034-P035 mixes requires only two MLPA reactions. In addition, P034/P035 requires the use of capillary sequencers and can detect duplications in both patients and DMD carriers. This SALSA kit is designed to detect deletions/duplications of one or more sequences in the aforementioned gene in a DNA sample. The DMD gene is located on the X-chromosome and almost all patients are male. Deletions of a s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding amplification product in males. Duplications can be detected by high quality agarose gel electrophoresis of A071-A078 amplification products (Johan den Dunnen & Stefan White, personal examination). Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak area, even when not located exactly on the ligation site! In addition, some 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 this gene is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA test. SALSA kits are sold by 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 kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA test kits includes a limited license to use these products for research purposes. The use of this SALSA 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). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA kit A071-A078 DMD Page 1 of 7

2 Related SALSA kits P034/P035 DMD: More s for DMD (capillary electrophoresis required) A012 ERBB2: Low-resolution detection of ERBB2 deletion/duplications References for SALSA kits A071-A078 DMD Schwartz M. et al. (2007). Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat Feb;28(2):205. White S.J. et al. (2006). Copy number variation in the genome; the human DMD gene as an example. Cytogenet Genome Res. 2006;115(3-4): Review. Lai K.K. et al. (2006). Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA). Clin Biochem Jan 11. Lalic T et al. (2005). Deletion & duplication screening in the DMD gene using MLPA. Eur J Hum Genet.13(11): Gatta V et al (2005). Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation amplification (MLPA). Hum Genet. Jun;117(1): Schwartz M and Duno M (2005). Multiplex ligation-dependent amplification is superior for detecting deletions/duplications in Duchenne muscular dystrophy. Clin Genet.; 67(2): Janssen B et al. (2005). MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics.; 6(1): Schwartz M and Duno M (2004). Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent amplification method. Genet Test.; 8(4): Data analysis The A071-A078 DMD mixes contain MLPA s with amplification products between 137 and 491 nt. In addition, they contain 5 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt and one synthetic ligation-dependent control fragment at 92 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. In some mixes, one or two reference s for other chromosome X sequences are also included. The PCR primers included with these kits do not contain fluorescent tags. Length differences between the amplification products of two s are at least 24 nt. The amplification products of the MLPA reactions can easily be separated by agarose gel electrophoresis. Analysis of samples can be done by simple visual examination of the ethidium bromide stained agarose gels, or in case of Agilent / Caliper microfluidics separation, by visual examination of the electrophoresis peak profiles. Examples of profiles obtained with the A071-A078 DMD mixes can be viewed on Analysis of the DMD gene is however much more simple with the use of mixes P034 and P035 combined with separation of amplification products by capillary electrophoresis as only two MLPA reactions are required instead of eight. Many copy number alterations in healthy individuals are described in the database of genomic variants: 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. These mixes were developed by J. Coffa & J.P. Schouten at. In case the results obtained with this mix lead to a scientific publication, it would be very much appreciated if the first mix designer could be made a coauthor. Info / remarks / suggestions for improvement: info@mlpa.com SALSA kit A071-A078 DMD Page 2 of 7

3 Table 1.a A071 - A074 DMD mixes A071 A Control fragment 2q14 92 Control fragment 144 Probe L01002 exon Probe L01690 reference (Xq11.2) 169 Probe L01005 exon Probe L01279 exon Probe L01008 exon Probe L01009 exon Probe L01010 exon Probe L01012 exon Probe L01013 exon Probe L01611 exon Probe L01332 reference(xq28) 299 Probe L01019 exon Probe L01020 exon Probe L01063 exon Probe L01612 exon Probe L01616 exon Probe L01520 exon Probe L01071 exon Probe L01072 exon Probe L01035 exon Probe L01036 exon Probe L01976 exon Probe L01975 DP427cAlt exon 1 A073 A Control fragment 2q14 92 Control fragment 2q Probe L01437 reference(xq28.1) 138 Probe L01041 exon Probe L01007 exon Probe L01004 exon Probe L01049 exon Probe L01609 exon Probe L01052 exon Probe L01050 exon Probe L01055 exon Probe L01014 exon Probe L01287 exon Probe L01574 exon Probe L01062 exon Probe L01572 exon Probe L01026 exon Probe L01281 exon Probe L01070 exon Probe L01069 exon Probe L01285 exon Probe L01033 exon Probe L01977 exon Probe L01978 exon 20 * Not ligation-dependent; these fragments indicate the amount of DNA used. These control fragments will not be visible on agarose gels. Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes. The identity of the genes detected by the reference s is available on request: info@mlpa.com. SALSA kit A071-A078 DMD Page 3 of 7

4 Table 1.b A075 - A078 DMD mixes A075 A Control fragment 2q14 92 Control fragment 2q Probe L01001 exon Probe L01615 exon Probe L01044 exon Probe L01571 exon Probe L01370 reference(2p16) 203 Probe L00465 reference(xp22.1) 226 Probe L01051 exon Probe L01011 exon Probe L01518 exon Probe L01054 exon Probe L01058 exon Probe L01057 exon Probe L01288 exon Probe L01021 exon Probe L01066 exon Probe L01065 exon Probe L01030 exon Probe L01283 exon Probe L01074 exon Probe L01073 exon Probe L01979 exon Probe L01980 exon 10 A077 A Control fragment 92 Control fragment 2q Probe L01042 exon Probe L01371 exon Probe L01608 exon Probe L01439 reference(xq28.1) 196 Probe L01610 exon Probe L00831 reference(xq22.3) 220 Probe L01450 reference(xq28.1) 242 Probe L01573 exon Probe L01333 reference(xq28) 266 Probe L01016 exon Probe L01060 exon Probe L01059 exon Probe L01024 exon Probe L01023 exon Probe L01068 exon Probe L01067 exon Probe L01533 exon Probe L01284 exon Probe L01076 exon Probe L01613 exon Probe L01981 exon Probe L01982 exon 30 * Not ligation-dependent; these fragments indicate the amount of DNA used. These control fragments will not be visible on agarose gels. Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes. The identity of the genes detected by the reference s is available on request: info@mlpa.com. SALSA kit A071-A078 DMD Page 4 of 7

5 Table 2. DMD s arranged according to chromosomal location Distance DMD MLPA Ligation site Partial sequence (24 nt adjacent DMD exon to next mix M18533 to ligation site) exon A L01975 Exon DP427c * GGCAGTAATAGA- ATGCTTTCAGGA kb A L01001 Exon TTCCCCCTACAG-GACTCAGATCTG kb A L01005 Exon TTCAAAAGAAAA-CATTCACAAAAT kb A L01009 Exon ACCTCTTCAGTG-ACCTACAGGATG 5.0 kb A L01013 Exon TGCCCTGAACAA-TGTCAACAAGGC 21.5 kb A L01574 Exon GTAGATGGAAAT-CATAAACTGACT 6.7 kb A L01021 Exon AACCAACAGTGA-AAAGATTCTCCT 7.0 kb A L01281 Exon CTGGAATAGTGT-GGTTTGCCAGCA kb A L01283 Exon CATTGAAGCCAT-CCAGGAAGTGGA 1.2 kb A L01033 Exon GCACAGGGATAT-GAGAGAACTTCT 52.8 kb A L01286 Exon GAAGACAAGTCA-TTTGGCAGTTCA 0.8 kb A L01041 Exon ATTTGACAGCCC-ATCAGGGCCGGG 29.9 kb A L01608 Exon AGAGCCTCTTGG-ACCTGATCTTGA 18.5 kb A L01049 Exon GTGGTAGTTGAT-GAATCTAGTGGA 22.0 kb A L01573 Exon ATGGGCAAACAT-CTGTAGATGGAC 0.2 kb A L01057 Exon CATGGCTTTCAG-AAAAAGAAGATG 7.8 kb A L01572 Exon AAGCAATCCATG-GGCAAACTGTAT 20.6 kb A L01065 Exon GGAACAGATCCT-GGTAAAGCATGC 27.2 kb A L01069 Exon AGAAGCTGTGTT-GCAGAGTCCTGA 16.3 kb A L01073 Exon AAAAGCTGAGAA-GTTCAGAAAACT 10.4 kb A L01978 Exon CGGTGGATCGAA-TTCTGCCAGTTG 6.4 kb A L01615 Exon GAAAGGACAAGG-ACCCATGTTCCT 12.7 kb A L01007 Exon TCAGGAGACCAT-GAGTGCCATCAG 3.6 kb A L01011 Exon GTGGCCTATACT-ATCTCAGCACCA 4.0 kb A L01518 Exon ATGGCCTGCCCT-TGGGGATTCAGA 1.1 kb A L01019 Exon GGCAGAAGATAA-AGAATGAAGCAG 8.8 kb A L01023 Exon CTGTAAGCCTCC-AGAAAGATCTAT 6.2 kb A L01616 Exon ACTGAGTCTGTA-AATAGTGTCATA 7.3 kb A L01284 Exon TTGGCATGAGTT-ATTGTCATACTT 3.0 kb A L01035 Exon GCACAGACCCTA-ACAGATGGCGGA 26.4 kb A L01039 Exon AAAAGTTGCTTG-AACAGAGCATCC 21.8 kb A L01371 Exon AAGGAGGCTGCC-CAAAGAGTCCTG 0.6 kb A L01609 Exon GCCTGCATTGGA-AACAAAGAGTGT 3.1 kb A L01051 Exon AGTCTGAAGTGG-AAATGGTGATAA 5.8 kb A L01055 Exon GCGAAAGGAAAT-GAATGTCTTGAC 15.5 kb A L01059 Exon CACCTGAAGAGT-ATCACAGAGGTA 0.5 kb A L01063 Exon ACATCACAAAGT-GGATCATTCAGG 1.8 kb A L01067 Exon 37 GACTCTACACGT-GACCAAGCAGCA 14.4 kb A L01071 Exon GGCTGAAATTCA-GCAGGGGGTGAA 2.4 kb A L01613 Exon GTTGCAAAGAGG-AGACAACTTACA 2.8 kb A L01079 Exon AAAAGGCTCTAG-AAATTTCTCATC 1.0 kb A L01002 Exon GAGGGCTTGTCT-GAGGATGGGGC 32.0 kb A L01279 Exon AACGATGATGGT-GATGACTGAAGA 22.6 kb A L01010 Exon GCATTGCAAAGT-GCAACGCCTGTG 70.6 kb A L01014 Exon GAACAGTTTCTC-AGAAAGACACAA kb A L01287 Exon ACAGATGCCAGT-ATTCTACAGGAA 36.3 kb A L01288 Exon AACATTGCTAGT-ATCCCACTTGAA 2.4 kb A L01026 Exon TCTCAAACAATT-AAATGAAACTGG 54.4 kb SALSA kit A071-A078 DMD Page 5 of 7

6 Distance DMD MLPA Ligation site Partial sequence (24 nt adjacent DMD exon to next mix M18533 to ligation site) exon A L01030 Exon CAGTTAAATCAT-CTGCTGCTGTGG 38.5 kb A L01285 Exon GGAAGAGATTTT-GTCTAAAGGGCA 16.7 kb A L01979 Exon GGTAAACCGTTT-ACTTCAAGAGCT 46.0 kb A L01042 Exon GCTCTGGCAGAT-TTCAACCGGGCT 44.4 kb A L01571 Exon Rev. CTAGCCTCTTGA-TTGCTGGTCTTG 50.2 kb A L01050 Exon CTGAGCAGGTCT-TAGGACAGGCCA 21.3 kb A L01054 Exon TGGCAGACAAAT-GTAGATGTGGCA 30.3 kb A L01058 Exon AAACTCATAGAT-TACTGCAACAGT kb A L01062 Exon AGTCCTGTTACA-AAGACGTTTGGA 10.5 kb A L01066 Exon CTGAAAGATGAT-GAATTAAGCCGG 17.8 kb A L01070 Exon TGAGACTGTACG-AATATTTCTGAC 0.8 kb A L01074 Exon TAGATGAGACCC-TTGAAAGACTCC 33.6 kb A L01977 Exon GCGCCTCTGAAA-GAGAACGTGAGC 96.0 kb A L01004 Exon AGGCAGCTGCAT-GAAGCCCACAG 25.0 kb A L01008 Exon GGTCCCTGGGAG-AGAGCCATCTCG 62.6 kb A L01012 Exon TCAAACAACTTG-CTGGGACCATCC 37.9 kb A L01016 Exon Rev. TTCTGCAGTCTT-CGGAGTTTCATG 13.4 kb A L01020 Exon CAGCTGCATGTG-ATGCCTTGGACC 3.0 kb A L01024 Exon TCCTGTCTTTTA-AAACTGGCATCA 2.6 kb A L01520 Exon Rev. GGACACTTGGCT-CAATGTTACTGC 21.1 kb A L01533 Exon TAGACTGGATGA-GACTGGAACCCC 2.4 kb A L01036 Exon GCTTTTTTTCTG-GTCGAGTTGCAA 1.7 kb A L01981 Exon ATCCCCGAATGG-GCTACCTGCCAG 0.8 kb A L01044 Exon ACTCTGATCAAC-TTCTGGCCAGTA 4.4 kb A L01610 Exon CCCCTCAGCTTT-CACACGATGATA 1.2 kb A L01052 Exon Rev. GCTATCATTTAG-ATAAGATCCATT 2.9 kb A L01611 Exon GCCCAGATCTTG-ATTTCCTTAGAG 22.1 kb A L01060 Exon GCTGAGCTCATT-GCTGAGGCCAAG 1.0 kb A L01612 Exon CTACCTCTCTAC-AGAGGTCCGACA 12.2 kb A L01068 Exon CCCCAGGACACA-AGCACAGGGTTA 7.5 kb A L01072 Exon CCTGGAAAGCCA-ATGAGAGAGGTT 4.8 kb A L01076 Exon TTCCACATGGCA-GATGATTTGGGC Total length of the DMD gene is kb!! The distance between different exons gives some indication about the likelihood that a single exon deletion/duplication occurs. It is e.g. unlikely that exon 35 is duplicated and exon 36 is not, as these exons are only 0.3 kb apart. In case you find a deletion/duplication of only one exon, we strongly recommend performing a sequencing analysis of this exon, as mutations / polymorphisms situated close to the ligation site can influence the peak area of the amplification product of that. Note: The exon numbering in Table 2 is different as compared to the exon numbering used by the NCBI in the NM_.. reference sequences! We used the same exon numbering as in all previous versions of this product description. The complete sequences are available on request. Please notify us of any mistakes: info@mlpa.com. SALSA kit A071-A078 DMD Page 6 of 7

7 kit A071-A78 DMD sample picture Figure 1. Agarose gel-electrophoresis analyzed with kits A071-A078 DMD (lot 0606) Implemented Changes compared to the previous product description version Version 06 (46) - Warning added below Table 2 that the exon numbering used is different from the NCBI exon numbering in the NM_ reference sequence. - New references added on page 2. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes throughout the document. - Various minor layout changes. - Control s have been renamed to reference s. SALSA kit A071-A078 DMD Page 7 of 7