SALSA MLPA probemix P125-B1 Mitochondria Lot B1-0216, B1-0312, B

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1 SALSA MLPA probemix P125-B1 Mitochondria Lot B1-0216, B1-0312, B Mitochondrial DNA (mtdna) differs from nuclear DNA in several ways. The complete mitochondrial genome is circular and only 16,569 bp long. Only a small number of genes needed for mitochondrial functions are encoded by the mtdna. Human cells contain an average of copies of the mitochondrial genome. Deletions in the mitochondrial DNA have been identified in various diseases including Pearson Syndrome, Kearns-Sayre Syndrome and Progressive external ophthalmoplegia. These deletions vary in size (1.3-8 kb) and location, but the most common deletion site is between positions 8469 and Diseases caused by mutations in mtdna are characterised by heteroplasmy: a mixture of wild-type and mutant mtdna is present in one cell. Usually, deletions in mtdna are detected when smaller fragments than expected are formed during PCR. Please note that identical deletions can cause different diseases depending on the tissue in which they occur. Because a deletion anywhere in the mitochondrial genome can also affect the transcription and translation of genes of which the sequence is intact, deletions of various sizes can cause similar phenotypes. Some tandem duplications of parts of the mitochondrial genome have also been described, as well as several mitochondrial point mutations resulting in a myopathy, such as MERRF and MELAS. This P125-B1 Mitochondria probemix contains probes for 32 different mtdna sequences that can be used to detect copy number changes. In addition, 5 mutation-specific probes specific for frequent mitochondrial point mutations, including the 3243A>G MELAS and the 8344A>G MERRF mutation, have been included. Due to the large difference in copy number between genomic and mitochondrial sequences, it is not possible to use reference probes targeting genomic sequences in this probemix. Instead, all 32 probes that detect a wild-type sequence are used as reference probes. In case of large deletions, many reference probes might be involved in the deletion and it may be necessary to reanalyse samples using a selection of these 32 probes. has not tested this probemix on patient samples. Based on literature, a minimum percentage of 23-40% mutant mtdna copies is needed to detect a deletion with this probemix (Mayorga et al. 2016, Tonska et al. 2012). The percentage heteroplasmy found will vary between tissues. The muscle biopsy is considered as the optimal material (Mayorga et al. 2016, Tonska et al. 2012, Kwon et al. 2011). The liver biopsy may be quite useful, while blood can be less informative in some cases (Tonska et al. 2012). Please note that the sensitivity of conventional PCR assays used for the detection of mitochondrial deletions in blood will be higher. However, the use of this MLPA assay may have advantages in some cases and may provide the location of suitable primers for a conventional PCR assay. Deletions of probe recognition sequences will be apparent by a reduced relative peak height of the amplification product of that probe. Apparent deletions of a single probe always require confirmation by other methods. We have no information on which percentage of defects in mtdna is caused by deletions and/or duplications. Since mitochondrial rrna is heavily expressed in each tissue, an RNAse treatment of samples is essential to obtain good MLPA results. Without RNAse treatment, the mitochondrial ribosomal RNA can bind to the template mtdna strand, thus competing with the MLPA probes. Since the degree of mtrna contamination varies between samples, some mitochondrial MLPA probes will yield irregular results when samples have not undergone RNAse treatment. On page 2, a possible RNAse treatment is described. 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 test probemixes and reagents includes a limited license to use these products for research purposes. SALSA P125 Mitochondria probemix Page 1 of 7

2 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 Acid Research 30, e57 (2002). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands References Mayorga L et al. (2016). Mitochondrial DNA deletions detected by Multiplex Ligation-dependent Probe Amplification. Mitochondrial DNA A DNA MappSeq Anal. 27: Tonska K et al. (2012). Molecular investigations of mitochondrial deletions: Evaluating the usefulness of different genetic tests. Gene. 506: Kwon et al. (2011). Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of mitochondrial DNA deletion in chronic progressive external ophthalmoplegia (CPEO). Ann Clin Lab Sci. 41: SD065 Binning DNA The SD065 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of 5 mutation-specific probes (266 nt probe L15252, 3243A>G mutation; 251 nt probe L14656, 3460G>A mutation; 239 nt probe L14658, 8344A>G mutation; 195 nt probe L15250, 11778G>A mutation; 231 nt probe L14655, 14484T>C mutation). SD065 consists of a plasmid that contains the sequences of all probes present in the P125-B1 probemix version. Inclusion of one reaction with 5 µl SD065 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signals, as for this purpose true mutation/snp positive patient samples or cell lines should be used. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD065 Binning DNA product description provided. This product is for research use only (RUO) Mitochondrial DNA The mutation rate in mtdna is about 10 times higher than in nuclear DNA, probably due to the lack of protective histones, an inferior repair system and the exposure to oxygen-free radicals generated by oxidative phosphorylation. The regions between nt and between nt are hypervariable. In addition, mtdna has no introns, meaning any random mutation will usually strike a coding sequence. Transcription of mtdna is polycistronic, which means that all genes encoded on the heavy and light strands are transcribed as 2 large precursor RNA strands. A deletion anywhere in the mitochondrial genome may affect the transcription or translation of genes of which the sequence is intact. Inherited anomalies of mtdna are all maternal as all mitochondria come from the ovum. Modifications MLPA protocol for mtdna 1. RNAse sample treatment The presence of RNA in the sample may result in a higher variability of some mtdna probes, in particular of probes detecting the RNR1 and RNR2 genes (142, 160, 178, 202 and 226 nt). This is due to the large amount of RNA for these genes that is expressed in mitochondria. An RNAse treatment is essential when performing MLPA on mitochondrial DNA. Please note that some automatic DNA purification methods (e.g. Roche Magnapure, Chemagen) do not include an RNAse treatment. The following method can be used to treat samples: Mix 4 µl sample DNA and 1 µl 0.5 mg/ml RNAse A. Incubate 30 minutes at 37 C. Continue with the 5 minutes 98 C DNA denaturation step of the MLPA protocol. SALSA P125 Mitochondria probemix Page 2 of 7

3 RNAse A is extremely stable; it can be diluted in TE and stored at -20 C. We recommend RNAse A7973 solution from Promega; use 4 mg/ml, diluted 8 fold in TE (1 ml is sufficient for ~8000 samples). Do not use more than the recommended amount. 2. Amount of mtdna used Reannealing of complementary strands could theoretically influence results when sequences are present in quantities larger than 100 copies per cell, as is usually the case in mitochondrial DNA. We therefore recommend using only 1-50 ng sample DNA. Although samples containing less sample DNA may still contain sufficient copies of mitochondrial DNA to yield good MLPA results, the sample DNA may be partly lost by adherence to tube walls etc. DNA samples containing 600 ng or more of human DNA, on the other hand, contain over 20,000,000 copies of mitochondrial DNA. When this much DNA is used, reannealing of sample DNA and increased viscosity of samples can yield more variable results. Data analysis The P125-B1 Mitochondria probemix contains 37 MLPA probes with amplification products between 136 and 427 nt. This includes 5 mutation-specific probes which will only generate a signal when the mutation is present. In addition, it contains 4 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt. These will only be visible when no sample DNA, or an extremely low amount of sample DNA is used. Data generated by this probemix should be normalised with a more robust method. (1) Intra-sample normalisation should be performed by dividing the signal of each probe by the signal of every other probe in that sample, thus creating as many ratios per probe as there are other 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. 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. 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 P125 Mitochondria probemix Page 3 of 7

4 Table 1. SALSA MLPA probemix P125-B1 Mitochondria Length (nt) SALSA MLPA probe * Q-fragments: DNA quantity; only visible when less than 1 ng human DNA has been used. 136 MT probe L ± RNR1 (MTRNR1) probe L COX1 (MTCO1) probe L ND4 (MTND4) probe L ± RNR1 (MTRNR1) probe L COX2 (MTCO2) probe L ND4 (MTND4) probe L ± RNR1 (MTRNR2) probe L COX2 (MTCO2) probe L ND5 (MTND5) probe L MT11778G>A probe L ± RNR2 (MTRNR2) probe L COX2 (MTCO2) probe L ATP6 (MTATP6) probe L ± RNR2 (MTRNR2) probe L ٨ MT14484T>C probe L ٨ MT8344A>G probe L reverse 247 ND5 (MTND5) probe L MT3460G>A probe L ATP6 (MTATP6) probe L reverse 266 MT3243A>G probe L reverse 286 ND1 (MTND1) probe L ATP6 (MTATP6) probe L ND6 (MTND6) probe L ND2 (MTND2) probe L COX3 (MTCO3) probe L CYTB (MTCYB) probe L ND2 (MTND2) probe L COX3 (MTCO3) probe L CYTB (MTCYB) probe L MTTC/Y probe L ND3 (MTND3) probe L CYTB (MTCYB) probe L COX1 (MTCO1) probe L ND4 (MTND4) probe L ND1 (MTND1) probe L ND5 (MTND5) probe L ± More variable. These probes are most sensitive to the presence of RNA in the DNA samples (see page 2). Mutation-specific probe. This probe will only generate a signal when the indicated mutation is present. It has been tested on artificial test DNA but not on positive human samples! ٨ Mutation-specific probe. The 231 and 239 nt mutation-specific probes give a 5-10% signal on wild type DNA. The height of the background peaks for these probes increases when the ligation period is longer than the recommended 15 minutes. The presence of the indicated mutations is most easily identified by a change in relative peak height as compared to other mutation-negative samples tested in the same experiment. Wild type sequence detected. The presence of the 8993T>G or 8993T>C mutation will result in a decreased probe signal. This probe is also sensitive to a G>A variant at position * Similar to most other MLPA probemixes, QDX2 control fragments are included in the P125-B1 probemix. Due to the high copy number of the mitochondrial DNA, the QDX2 peaks at 88, 92, 96, 100 and 105 nt will usually not be visible. + This probe can give a lower signal in the presence of ionic contaminants due to the formation of quadruplexes. Repeating the experiment after an extra purification step is recommend to reduce the amount of contaminants. Please notify us about any mistakes: info@mlpa.com. Ligation site of the probe on the mitochondrial genome SALSA P125 Mitochondria probemix Page 4 of 7

5 Table 2. P125 Mitochondria probes arranged according to location Length (nt) SALSA MLPA probe Gene / mutation Location / Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe 142 ± L03649 RNR TAAATCACCACG-ATCAAAAGGGAC 349 nt 160 ± L15249 RNR ATACCCCACTAT-GCTTAGCCCTAA 598 nt 178 ± L03651 RNR ACCTAGCCCCAA-ACCCACTCCACC 1005 nt 202 ± L04299 RNR GACCTGCCCGTG-AAGAGGCGGGCA 440 nt 226 ± L03653 RNR CAAGAGAAATAA-GGCCTACTTCAC 109 nt L A>G rev. GATTACCGGGCC-CTGCCATCTTAA 217 nt L G>A CCTTCGCTGACA-CCATAAAACTCT 191 nt L14661 ND GCCGTTTACTCA-ATCCTCTGATCA 453 nt L03646 ND GACCCTACTTCT-AACCTCCCTGTT 752 nt L15460 ND ATCCGGCCTGCT-TCTTCTCACATG 571 nt L03648 ND CAGTTTGAACAT-ACAAAACCCACC 395 nt L03657 MTTC/Y AAAATCACCTCG-GAGCTGGTAAAA 200 nt L15256 COX TATTCGAGCCGA-GCTGGGCCAGCC 1124 nt L03644 COX AAAATCCATTTC-ACTATCATATTC 503 nt L03654 COX GAAGAGCTTATC-ACCTTTCATGAT 263 nt L03655 COX CTGAACCTACGA-GTACACCGACTA 292 nt L03656 COX AACCACAGTTTC-ATGCCCATCGTC 139 nt 239 ٨ L A>G rev. AGAGGTGTTGGC-TCTCTTAATCTT 313 nt L03658 ATP AACCGACTAATC-ACCACCCAACAA 336 nt L T rev. GGCGTACGGCCA-GGGCTATTGGTT 177 nt L03659 ATP GCCTACGTTTTC-ACACTTCTAGTA 157 nt L03661 COX CTTCCACTCCAT-AACGCTCCTCAT 589 nt L03662 COX TTTGGCTTCGAA-GCCGCCGCCTGA 256 nt L15255 ND CCACCCCTTACG-AGTGCGGCTTCG 751 nt L15257 ND AGCTGTTCCCCA-ACCTTTTCCTCC 606 nt L03668 ND CTGACAAAACAC-ATAGCCTACCCC 250 nt L G>A CACTCACAGTCA-CATCATAATCCT 290 nt L03669 ND GAAAACACCCTC-ATGTTCATACAC 407 nt L03670 ND ACCTTTATTATC-AGTCTCTTCCCC 692 nt L14662 ND CAAACTCTAACA-CTATGCTTAGGC 855 nt L03672 ND TAGAAAAGCTAT-TACCTAAAACAA 462 nt 231 ٨ L T>C AAAGACAACCAC-CATTCCCCCTAA 76 nt L15459 ND AATAACACACCC-GACCACACCGCT 265 nt L15253 CYTB ACCCCATCCAAC-ATCTCCGCATGA 517 nt L15254 CYTB TCCACCTCCTAT-TCTTGCACGAAA 422 nt L03665 CYTB ACCTGAATCGGA-GGACAACCAGTA 656 nt L14663 MT TCCGTGAAATCA-ATATCCCGCACA 891 nt 142 ± L03649 RNR TAAATCACCACG-ATCAAAAGGGAC 349 nt 160 ± The mitochondrial DNA is circular and only nt long ± More variable. These probes are most sensitive to the presence of RNA in the DNA samples (see page 2). Mutation-specific probe. This probe will only generate a signal when the indicated mutation is present. It has been tested on artificial test DNA but not on positive human samples! ٨ Mutation-specific probe. The 231 and 239 nt mutation-specific probes give a 5-10% signal on wild type DNA. The height of the background peaks for these probes increases when the ligation period is longer than the recommended 15 minutes. The presence of the indicated mutations is most easily identified by a change in relative peak height as compared to other mutation-negative samples tested in the same experiment. Wild type sequence detected. The presence of the 8993T>G or 8993T>C mutation will result in a decreased probe signal. This probe is also sensitive to a G>A variant at position This probe can give a lower signal in the presence of ionic contaminants due to the formation of quadruplexes. Repeating the experiment after an extra purification step is recommend to reduce the amount of contaminants. Note: Complete probe sequences are available on Please notify us of any mistakes: info@mlpa.com. SALSA P125 Mitochondria probemix Page 5 of 7

6 SALSA MLPA probemix P125-B1 Mitochondria sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P125-B1 Mitochondria (lot B1-0216). Please note that the 231 and 239 nt mutation-specific probes generate a 5-10% background signal on mutation-negative samples. Figure 2. Capillary electrophoresis pattern of SD065 sample DNA (approximately 50 ng) analysed with SALSA MLPA probemix P125-B1 Mitochondria (lot B1-0216). The locations of the mutation-specific probes at 195, 231, 239, 251 and 266 nt are indicated. SALSA P125 Mitochondria probemix Page 6 of 7

7 Implemented Changes compared to the previous product description versions. Version October 2018 (55) - Warnings added to Table 1 and 2, 328 nt probe L15253, 337 nt probe L03648, and 409 nt probe L Warning adjusted in Table 1 and 2, 260 nt probe L Version June 2017 (55) - Information about heteroplasmy and the optimal tissue to use added on page 1. - Recommended amount of sample DNA adjusted on page 3. - New reference added on page 2. Version August 2016 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new pictures included). - Information about SD065 Binning DNA added on page 2. - New reference added on page 2. - Various minor textual changes throughout the document. Version July 2015 (54) - Figure based on the use of old MLPA buffer (replaced in December 2012) removed. - New reference added. - Various minor textual changes throughout the document. SALSA P125 Mitochondria probemix Page 7 of 7