MRC-Holland MLPA. Related SALSA MLPA probemix P091 CFTR: contains probes for the CFTR gene, related to chronic pancreatitis.

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1 SALSA MLPA probemix P242-B3 Pancreatitis Lot B As compared to version B2 (lot B2-1212), one flanking probe has been removed and four reference probes have been replaced. Hereditary Pancreatitis (HP) is a rare genetic condition characterised by recurrent episodes of pancreatic attacks, which can progress to chronic pancreatitis. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. The PRSS1 gene (5 exons) spans ~3.6 kb of genomic DNA and is located on chromosome 7q34, about 142 Mb from the p-telomere. The SPINK1 gene (4 exons) spans ~7.1 kb of genomic DNA and is located on chromosome 5q32, about 147 Mb from the p-telomere. The P242-B3 Pancreatitis probemix contains probes for each exon of PRSS1, including two probes for exon 1. Furthermore, it contains one upstream and two downstream probes on 7q34. Four probes are included for the SPINK1 gene, detecting the first three exons and a region downstream of exon 4, no probe detecting exon 4 is included. Finally, probemix contains ten reference probes detecting several different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes 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 this gene are expected to be small (point) mutations which will not be detected by this SALSA test. SALSA probemixes and reagents are sold by 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 this 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 Acid Research 30, e57 (2002). Related SALSA probemix P091 CFTR: contains probes for the CFTR gene, related to chronic pancreatitis. Reference Rygiel A.M. et al. (2015) Gene conversion between cationic trypsinogen (PRSS1) and the pseudogene trypsinogen 6 (PRSS3P2) in patients with chronic pancreatitis. Human Mutation. 36: More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P242 Pancreatitis probemix Page 1 of 5

2 Data analysis The P242-B3 Pancreatitis probemix contains 23 MLPA probes with amplification products between 135 and 346 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 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 exons 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, can be downloaded free of charge from our website 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 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. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P242 Pancreatitis probemix Page 2 of 5

3 Table 1. SALSA MLPA P242-B3 Pancreatitis probemix Length Chromosomal position SALSA MLPA probe (nt) reference SPINK1 PRSS1 region 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 135 Reference probe L p PRSS1 probe L09483 Exon Reference probe L q BRAF probe L03624 Upstream 174 PRSS1 probe L09484 Exon SPINK1 probe L08297 Exon Reference probe L q CASP2 probe L28279 Downstream 223 SPINK1 probe L28280 Exon * Reference probe L p PRSS1 probe L09361 Exon SPINK1 probe L28281 Downstream 256 Reference probe L p PRSS1 probe L10056 Exon * Reference probe L p Reference probe L q SPINK1 probe L28282 Exon PRSS1 probe L28284 Exon Reference probe L p CASP2 probe L01583 Downstream 328 * Reference probe L q PRSS1 probe L09482 Exon * Reference probe L q13 * New in version B3 (from lot B onwards). Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P242 Pancreatitis probemix Page 3 of 5

4 Table 2. P242 probes arranged according to chromosomal location Table 2a. PRSS1 region Length SALSA PRSS1 Ligation site Partial sequence (24 nt Distance to (nt) MLPA probe Exon NM_ adjacent to ligation site) next probe L03624 BRAF gene TTGGGACACTGA-TATTTCCTGGCT kb Start codon (ex 1) L10056 Exon 1 32 nt before ex 1 CTGGATCCTCGT-GAGGTATAAAGA 0.1 kb L28284 Exon TGGCAGCTGCTC-GTGAGTATCATG 1.2 kb L09482 Exon CTCCCTCATCAA-CGAACAGTGGGT 1.2 kb L09361 Exon GACATCATGTTA-ATCAAGCTCTCC 0.7 kb L09483 Exon GGCAAGGATTCA-TGTCAGGTGATT 0.4 kb L09484 Exon ACAACTATGTGA-AATGGATTAAGA kb Stop codon (ex 5) L01583 CASP2 gene TTTCTTACAGTT-GAGCTGTGACTA 11.2 kb L28279 CASP2 gene TGGGGTTGACCA-ACAAGATGGAAA Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. SPINK1 Length (nt) SALSA MLPA probe SPINK1 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe Start codon (ex 1) L28280 Exon GCGGTGCAGTTT-TCAACTGACCTC 2.0 kb L28282 Exon TATTTCTAGGTA-CACTGGAGCTG 1.5 kb L08297 Exon GCACCAAGATAT-ATGACCCTGTCT 4.1 kb No Probe Exon 4 Stop codon (ex 4) L28281 Downstream 577 nt after ex 4 CTTAGCCTTAGT-TCTTCCCCAGCA The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P242 Pancreatitis probemix Page 4 of 5

5 SALSA MLPA probemix P242-B3 Pancreatitis sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P242-B3 Pancreatitis (lot B3-0215). Implemented Changes compared to the previous product description versions. Version April 2015 (54) - Product descriptions adapted to a new product version (version number changed, lot number changed, changes in Table 1 and 2, new picture included). - New reference added on page 1. Version 08 (49) - This product description has been changed to incorporate a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Version 07 (48) - Warning added in Table 1, 256 nt probe L Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (47) - Remark on RefSeqGene standard added below Table 2. - Small correction of chromosomal locations in Table 1 and 2. Version 04 (46) - Various minor layout changes. - Various minor textual changes on page 1 and 2. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Ligation sites of the probes targeting the PRSS1 gene updated according to the updated NM_reference sequence. SALSA P242 Pancreatitis probemix Page 5 of 5