SALSA MLPA Probemix P014-B1 Chromosome 8 Lot B and B

Transcription

1 SALSA MLPA Probemix P014-B1 Chromosome 8 Lot B and B Copy number changes of the human chromosome 8 are common in many types of tumours. In most cases, losses of 8p sequences and gains of 8q sequences are detected. This probemix is designed to have probes evenly spread over chromosome 8 and to contain probes for tumour suppressors (e.g. MTUS, LZTS1), DNA damage response genes (e.g. MCPH1) and cell cycle genes (e.g. MYC). For a complete list of chromosome 8 probes in this product, see Table 2a. This probemix contains 44 probes for different genes on chromosome 8 that are suggested to be involved in human tumours. In addition, 12 reference probes have been included in this probemix, detecting 12 different autosomal chromosomal locations, which are relatively silent in most tumour types. However, it should be noticed that tumour karyotypes can harbour multiple numerical and structural aberrations, which can complicate interpretation of these reference probes. This SALSA 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. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in these genes are expected to be small (point) mutations, which will not be detected by this SALSA test. This SALSA MS- probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes. The use of a SALSA 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 Acids Research 30, e57 (2002). Related SALSA probemixes P175 Tumour gain: Contains probes for the following genes: MDM4, MYCN-ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, BRAF/BRAF V600E mutation, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2-TOP2A, AURKA and AR. P294 Tumour loss: Contains probes for the following genes and chromosomal regions: 1p36, VHL, FHIT, APC, CDKN2A-CDKN2B, PTCH, TSC1, PTEN, WT1, BRCA2, RB1-MIR15, TSC2, TP53, NF1, BRCA1, SMAD4, STK11, FKBP8, SMARCB1 and FAM123B. References for SALSA P014 Chromosome 8 mix Yong ZWE et al (2014) Genetic alterations of chromosome 8 genes in oral cancer. Sci Rep. 4:6073. Passariello A et al (2013) Constitutional 11q14-q22 chromosome deletion syndrome in a child with neuroblastoma MYCN single copy. Eur J Med Genet. 56: Rumiato E et al (2011) DNA copy number profile discriminates between oesophageal adenocarcinoma and squamous cell carcinoma and represents an independent prognostic parameter in esophageal adenocarcinoma. Cancer Lett. 310: Kastler S et al (2010) POU5F1P1, a putative cancer susceptibility gene, is overexpressed in prostatic carcinoma. Prostate. 70: Bremmer JF (2005) A noninvasive genetic screening test to detect oral preneoplastic lesions. Lab Invest. 85: Buffart TE et al (2005) DNA copy number changes at 8q11-24 in metastasized colorectal cancer. Cell Oncol. 27: SALSA probemix P014 Chromosome 8 Page 1 of 6

2 More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Data analysis The P014-B1 Chromosome 8 probemix contains 56 MLPA probes with amplification products between 123 and 504 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA Denaturation control fragments (D-fragments) at nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix should be normalised with a more robust method, as the target sites of the reference probes maybe gained or lost. (1) Intra-sample normalisation should be performed by dividing the signal of each target-specific probe by the signal of every single reference probe in that sample, thus creating as many ratios per target-specific probe as there are reference probes. Subsequently, the median of all these produced ratios per probe should be taken; this is the probe s Normalisation Constant. (2) Secondly, inter-sample comparison should be performed by dividing the Normalisation Constant of each probe in a given sample by the average Normalisation Constant of that probe in all the reference samples. Data normalisation should be performed within one experiment. Always use sample and reference DNA extracted with the same method and derived from the same source of tissue. Confirmation of deletions, duplications and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Warning: MLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. Gains or losses of genomic regions or genes may not be detected if the percentage of tumour cells is low. Furthermore, although reference probes are located in silent regions that are not frequently altered in copy number in most tumour types, there is always a possibility that one or more reference probes do show a copy number alteration in a sample. Normal copy number variation in healthy individuals is described in the database of genomic variants: When in doubt, users should always verify the latest updates of the database and scientific literature when interpreting their findings. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P014 Chromosome 8 Page 2 of 6

3 Table 1. SALSA MLPA P014-B1 Chromosome 8 probemix Length Chromosomal position SALSA MLPA probe (nt) Reference Chr. 8p Chr. 8q MV Location (hg18) 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 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 123 Reference probe L q Reference probe L q RIPK2 probe L q PPP2R2A probe L p GATA4 probe L p Reference probe L q LPL probe L p MYC probe L q «KLF10 probe L q NCOA2 probe L q MYBL1 probe L q Reference probe L q MYC probe L q TCEB1 probe L q TPD52 probe L q «RECQL4 probe L q NEFL probe L p Reference probe L p SLA probe L q MCPH1 probe L p TNFRSF10B probe L p MOS probe L q PTP4A3 probe SP0531-L q «ZNF703 probe L p Reference probe L p E2F5 probe L q CHD7 probe L q RNF139 probe L q MTDH probe L q ASAP1 probe L q Reference probe L q LZTS1 probe L p WHSC1L1 probe L p PTK2 probe L q PRKDC probe L q RRM2B probe L q EXT1 probe L q NKX3-1 probe L p Reference probe L p NRG1 probe L p FGFR1 probe L p KHDRBS3 probe L q KCNQ3 probe L q EBAG9 probe L q Reference probe L p NDRG1 probe L q DLGAP2 probe L p EIF3H probe L q Reference probe L q PRDM14 probe L q MTUS1 probe L p WISP1 probe L q * RAD54B probe L q RHOBTB2 probe L p SALSA probemix P014 Chromosome 8 Page 3 of 6

4 Length Chromosomal position SALSA MLPA probe (nt) Reference Chr. 8p Chr. 8q MV Location (hg18) 490 Reference probe L p Reference probe L p «This probe is located within, or close to, very strong CpG islands. 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: Please notify us of any mistakes: info@mlpa.com. The identity of the genes detected by the reference probes is available in Table 2b. Table 2a. Chromosome 8 probes arranged according to chromosomal location Length SALSA MLPA Partial sequence Chromosomal Distance to Gene (nt) probe (24 nt adjacent to ligation site) position next probe L05848 DLGAP2 GTGAAGTTGAAT-CTCAGGCCATGG 8p kb L25275 MCPH1 GCAAATACTCAT-TAGACTACCTTA 8p kb L20557 GATA4 AAGAACCTGAAT-AAATCTAAGACA 8p kb L25269 MTUS1 GCCTCGCTACTT-TCTGCAGCTTGT 8p kb L21358 LPL TTTCCTCCCCAA-CAGTCTTCCATT 8p kb L25663 LZTS1 GCTGCAGCGCAA-GAAGAACGAGGC 8p kb L24843 RHOBTB2 GTTGCATTTGTT-TTGTGGGCATTT 8p kb L21781 TNFRSF10B GGTGATTGTACA-CCCTGGAGTGAC 8p kb L25330 NKX3-1 CCTGTGTCACTG-AATATCAACTCT 8p kb L25272 NEFL GGAACAAGCAGC-TAAGAAGAAAGA 8p kb L25326 PPP2R2A AAGCCTCGCACA-GTTCTGAAGCCT 8p kb L25331 NRG1 AAAACTTTCTGT-GTGAATGGAGGG 8p kb 261 «17595-L25667 ZNF703 CACTTTGGGCCT-AAGCCGGTACCA 8p kb L25662 WHSC1L1 AAGATTACTGTT-TTCAATGTGGAG 8p kb L00624 FGFR1 CAACCTCTAACT-GCAGAACTGGGA 8p kb L25660 PRKDC GGTGAAGTTCAT-CCTAGTGAGATG 8q kb L00621 MOS CTGTGGCCATAA-AGCAAGTGAACA 8q kb L25327 CHD7 AGGAACAGACAT-GCTAGCAGATGG 8q kb L12249 MYBL1 CTGTTGACTGAA-GACATTTCAGAC 8q kb L12830 PRDM14 CACTCTGGAGAC-AGACCATACCAG 8q kb L10399 NCOA2 GCGTGGCAAGAA-GAGTTCCCATGA 8q kb L22651 TCEB1 ATGGCATCAGGT-CCTTCACAGCCA 8q kb L00620 TPD52 CTTTGTTCTGCT-ACCCACTGCCAG 8q kb L00617 E2F5 GCACCTTCTGGT-ACACAACTGGAG 8q kb L25271 RIPK2 GCTTGGCCATTG-AGATTTCGCATC 8q kb L24842 RAD54B GTCCAAGGAACA-AGAAGAGAAATC 8q kb L23263 MTDH ACCTCAAAGTGT-AACAGCAAAGCA 8q kb L15225 RRM2B GCTTTCAAATTC-TCATCGAGAATG 8q kb 166 «19026-L24836 KLF10 AGAAAAGTGATT-TTGAAGCTGTAG 8q kb L24839 EBAG9 CCACCATGGCCA-TCACCCAGTTTC 8q kb L25659 EIF3H TTCACTGCCCAA-AACTTAGGCAAG 8q kb L02480 EXT1 TCCCAGTGCAGA-TCCACTCATCAG 8q kb L16047 RNF139 ATCATCGACGCC-ATCTTCAACTCC 8q kb L21679 MYC AACAACCGAAAA-TGCACCAGCCCC 8q kb L22108 MYC GAACGAGCTAAA-ACGGAGCTTTTT 8q kb L18499 ASAP1 TTCCTTTCAGGC-TGTCCTTCGATG 8q kb L06164 KCNQ3 AGACCATCCACA-TCAGAAATCGAA 8q kb L25274 SLA TCAGCTATGGCC-TTCGAGAGAGCA 8q kb L24841 WISP1 GATCTTGCCTAA-ATCCCAAGAAAT 8q kb L25658 NDRG1 CCAGGAAGCATT-TCAGCCAGCTGA 8q kb L00622 KHDRBS3 TTCCATCTCAAT-GATGATCTCCAT 8q kb L25661 PTK2 CTGGTTCACATG-GAATCACAGCCA 8q kb SP0531- AGAGGTTGCCTA-40nt spanning oligo- PTP3A4 L25332 GTGAGGCTGTGG 8q kb 211 «01036-L00609 RECQL4 AGGCAAACGTTT-TCAAAACCTCGA 8q SALSA probemix P014 Chromosome 8 Page 4 of 6

5 «This probe is located within, or close to, very strong CpG islands. 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: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. Table 2b. P014 reference probes arranged according to chromosomal location Length SALSA MLPA Partial sequence MV location Gene Location (nt) probe (24 nt adjacent to ligation site) (HG18) L03872 SLC2A1 1p34 GCCATCTTTTCT-GTTGGGGGCATG , L19465 PEX13 2p15 TGAGGATGACCA-TGTAGTTGCCAG , L06145 SCN2A 2q24 TGGTCAACAACT-ACAGTGAGTGCA , L18705 RAB7A 3q21 CACAATAGGAGC-TGACTTTCTGAC , L13645 IL4 5q31 ATCGACACCTAT-TAATGGGTCTCA , L11263 PKHD1 6p12 TCACAGCTGGTT-TCCTGAAAGGCT , L25273 DNAI1 9p13 AGGTGATTTCAG-AAACAGGAAACC , L25279 ZNF25 10p11 CAGGTGATTCCT-GGGGCTGCCAGC , L23200 RPGRIP1 14q11 GCTCAGGGCAGA-GCTGAAGGAAGA , L21570 SPG11 15q15 GTCAAAGGATTC-CCCGTTACTACT , L17529 RNMT 18p11 TACAATGAACTT-CAGGAAGTTGGT , L25270 KCNJ6 21q22 CTCGAAGCTCCT-ACATCACCAGTG , Note: Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA MLPA Probemix P014-B1 Chromosome 8 sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA probemix P014-B1 Chromosome 8 (lot B1-0916). Implemented Changes compared to the previous product description versions. Version June 2017 (T08) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2a/b, new picture included SALSA probemix P014 Chromosome 8 Page 5 of 6

6 - New references added on page 1 - Small changes of probe lengths in Tables 1 and 2a/b: in order to better reflect the true lengths of the amplification products. Version March 2016 (T08) - Various minor textual and layout changes. - New reference added on page 2. Version 08 (52) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Various textual changes on page 1. - New reference was added on page 1. SALSA probemix P014 Chromosome 8 Page 6 of 6