Related SALSA MLPA kits P190 CHEK2: Breast cancer susceptibility, genes included: CHEK2, ATM, BRCA1&2, PTEN, TP53

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1 SALSA MLPA KIT P056-A2 TP53 Lot One reference probe has been replaced, five probes have a small change in length (but no change in sequence detected) and four extra control fragments at nt have been included. LI-FRAUMENI SYNDROME (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome. LFS is characterized by autosomal dominant inheritance and early onset of tumors, multiple tumors within an individual, and multiple affected family members. Approximately 70% of LFS cases and 40% of LFL (Li- Fraumeni like syndrome) cases contain germline mutations in the p53 gene on chromosome 17p13.1. In a subset of non-p53 patients with LFS, mutations have been identified in the CHEK2 gene on chromosome 22q11. A third LFS locus has been identified on chromosome 1q23. This P056-A2 TP53 probemix contains probes for each of the 11 exons of the TP53 gene on 17p13.1, as well as several probes at close distances telomeric and centromeric of the TP53 gene. Two probes detect sequences in exon 1 of TP53. In addition, two probes are present for the CHEK2 gene on chromosome 22q12.1, as well as one probe specific for the CHEK2 1100delC mutation. This probe will only give a signal when the mutation is present! Please note more CHEK2 probes are available in SALSA MLPA kit P190 CHEK2. Eleven reference probes for sequences located on other chromosomes are also included. This SALSA MLPA kit is designed to detect deletions/duplications of one or more exons of the TP53 gene. Heterozygote deletions of probe recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. However, mutations and/or polymorphisms very close to the probe ligation site may also result in a reduced relative peak area. Therefore, apparent deletions detected by a single probe always require confirmation by other methods. Please note that most defects in this gene are expected to be small (point) mutations, most of which will not be detected by this MLPA 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 MLPA kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA MLPA test kits includes a limited license to use these products for research purposes. The use of this SALSA MLPA 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). Related SALSA MLPA kits P190 CHEK2: Breast cancer susceptibility, genes included: CHEK2, ATM, BRCA1&2, PTEN, TP53 More information Website : info@mlpa.com Fax : Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA kit P056 TP53 Page 1 of 5

2 Data analysis The P056 TP53 probemix contains 37 MLPA probes with amplification products between 127 and 490 nt. The 490 nt probe will only be visible in samples with the CHEK2 1100delC mutation. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Qfragments) 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 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. When only small numbers of samples are tested, visual comparison of peak profiles should be sufficient to easily identify exon deletions. Comparison of results should preferably 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, can be downloaded free of charge from our website CHEK2 1100delC probe This P056 probemix contains a CHEK2 mutation-specific 1100delC probe. Results obtained with this CHEK2 1100delC probe should be treated with caution! We have received reports of experiments at three different laboratories in which the CHEK2del1100 peak in the P045 BRCA2 probemix spuriously appeared in known normal controls as well as in all other samples. We have observed the same problem with the similar CHEK2 probe in this P190 CHEK2 probemix. The cause of this phenomenon is unknown and despite several attempts, we were not able to reproduce the results. In case the peak of the 1100delC probe is observed at 490 nt, always confirm the result by sequencing. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA kit P056 TP53 Page 2 of 5

3 SALSA MLPA P056-A2 TP53 probemix Length Chromosomal position SALSA MLPA probe (nt) reference other 17p 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 127 Reference probe 0797-L q EFNB3 probe 3962-L MPDU1 probe 6143-L EFNB3 probe 1984-L Reference probe 1063-L q TP53 probe 1588-L06028 Exon POLR2A probe 6146-L Reference probe 1217-L q TP53 probe 1996-L01536 Exon TP53 probe 2375-L01497 Exon * Reference probe 2333-L q PMP22 probe 1461-L p Reference probe 2873-L p TP53 probe 2376-L01498 Exon TP53 probe 2377-L01499 Exon CHEK2 probe 2578-L02040 promotor 274 TP53 probe 2378-L01500 Exon TP53 probe 1999-L01158 Exon SHBG probe 6145-L TP53 probe 0709-L00458 Exon TP53 probe 1991-L13860 Exon Reference probe 1789-L q Reference probe 1042-L q Reference probe 1774-L q ATP1B2 probe 2373-L TP53 probe 0345-L00171 Exon 12 ** 355 Reference probe 1920-L q NF2 probe 1580-L q Reference probe 0655-L q ATP1B2 probe 3961-L TP53 probe 1587-L01159 Exon 11 ** 400 CHEK2 probe 2579-L12282 Exon TP53 probe 2263-L01749 Exon Reference probe 1842-L p POLR2A probe 6147-L MPDU1 probe 6144-L CHEK2 probe 1772-L delC mut specific * New in version A2 (from lot 1108 onwards) Small change in length as compared to previous lots. No change in sequence detected. Results obtained with this CHEK2 1100delC probe should be treated with caution! Please read the note on page 2. This probe will only generate a signal when the CHEK2 point mutation is present. ** See note on the exon numbering on page 4. 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 probes is available on request: info@mlpa.com. SALSA kit P056 TP53 Page 3 of 5

4 P056 17p probes arranged according to chromosomal location Length (nt) SALSA MLPA probe Gene / Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe centromeric L00926 PMP22 TTAACATCCCTT-GCATTTGGCTGC Kb L06045 EFNB3 gene, exon 2 TCTCCTAATTAT-GAGTTCTACAAG 2.5 Kb L06047 EFNB3 gene, exon 1 GTCCGGGCTGAA-GAGCCAGGCAGC 17.9 Kb start codon L01749 TP53 exon nt before start codon CTTCCTCCGGCA-GGCGGATTACTT 0.2 Kb L06028 TP53 exon TCCGGGGACACT-TTGCGTTCGGGC 10.8 Kb L01536 TP53 exon CTCTTGCAGCAG-CCAGACTGCCTT 0.2 Kb L01497 TP53 exon TTCCTGAAAACA-ACGTTCTGGTAA 0.1 Kb L00458 TP53 exon CATCTACAGTCC-CCCTTGCCGTCC 1.1 Kb L01498 TP53 exon CAAGATGTTTTG-CCAACTGGCCAA 0.3 Kb L01499 TP53 exon TGGATGACAGAA-ACACTTTTCGAC 0.7 Kb L01500 TP53 exon CGGCATGAACCG-GAGGCCCATCCT 0.4 Kb L01158 TP53 exon CTGTCCTGGGAG-AGACCGGCGCAC 0.2 Kb L13860 TP53 exon ACTGCCCAACAA-CACCAGCTCCTC 2.9 Kb L01159 TP53 exon TTCCGAGAGCTG-AATGAGGCCTTG 1.0 Kb L00171 TP53 exon AAAGGGTCAGTC-TACCTCCCGCCA 13.9 Kb Stop codon L03348 ATP1B2 gene, exon 7 GTGAATGTAGAA-TGTCGCATCAAC 4.3 Kb L13861 ATP1B2 gene, exon 1 CCGCGCCACCAA-GATGGTCATCCA 19.6 Kb L13859 SHBG, exon 6 TGGGCCTTCTCT-TTGGACCTGGGA 44.4 Kb L06050 MPDU1, exon 7 AAAAGGCGCAGT-AGAGCCAGCTAC 1.5 Kb L05689 MPDU1, exon 3 TCTCCAGTCTGT-AATGCTGGAGCT 77.0 Kb L05689 POLR2A, exon 21 GCATACAACGTT-GAGGAAGGTGAC 7.4 Kb L05690 POLR2A, exon 14 GCGGCAGACGTT-TGAGAATCAGGT telomeric Note: Exon numbering of the TP53 gene has changed as compared to previous versions of this product description (description version 1-6). We have now adopted the exon numbering mentioned in the Genbank NM_ file. As a result, what was probes which were previously described as targeting exon 10 and 11 now recognise exon 11 and 12, respectively. This exon numbering might be different as compared to (older) literature! No probe is present for exon 10, which is not present in the NM_ transcript. Exon 10 is only present in some alternative transcripts encoding the p53gamma variant protein. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA kit P056 TP53 Page 4 of 5

5 SALSA MLPA kit P056-A2 TP53 sample picture Dye Signal Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P056-A2 TP53 (lot 1108). SALSA kit P056 TP53 Page 5 of 5