SALSA MLPA probemix P125-B1 Mitochondria Lot B1-0216, B1-0312, B1-0609. 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 300-400 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 13147. 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
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 : www.mlpa.com E-mail : 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. 27:2864-7. Tonska K et al. (2012). Molecular investigations of mitochondrial deletions: Evaluating the usefulness of different genetic tests. Gene. 506:161-5. 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. 41:385-9. 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 04904-L15252, 3243A>G mutation; 251 nt probe 13307-L14656, 3460G>A mutation; 239 nt probe 13309-L14658, 8344A>G mutation; 195 nt probe 13305-L15250, 11778G>A mutation; 231 nt probe 13306-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 45-287 nt and between 16105-16348 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
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 64-70-76-82 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 www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. 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
Table 1. SALSA MLPA probemix P125-B1 Mitochondria Length (nt) SALSA MLPA probe 64-70-76-82* Q-fragments: DNA quantity; only visible when less than 1 ng human DNA has been used. 136 MT probe 13314-L14663 16420-16421 142 ± RNR1 (MTRNR1) probe 04293-L03649 742-743 148 COX1 (MTCO1) probe 04288-L03644 7146-7147 154 ND4 (MTND4) probe 04312-L03668 11528-11529 160 ± RNR1 (MTRNR1) probe 13311-L15249 1091-1092 166 COX2 (MTCO2) probe 04298-L03654 7649-7650 171 ND4 (MTND4) probe 04313-L03669 12068-12069 178 ± RNR1 (MTRNR2) probe 04295-L03651 1689-1690 184 COX2 (MTCO2) probe 04299-L03655 7912-7913 190 ND5 (MTND5) probe 04314-L03670 12475-12476 195 MT11778G>A probe 13305-L15250 11778-11779 202 ± RNR2 (MTRNR2) probe 04296-L04299 2694-2695 211 COX2 (MTCO2) probe 04300-L03656 8204-8205 220 ATP6 (MTATP6) probe 04905-L03658 8656-8657 226 ± RNR2 (MTRNR2) probe 04472-L03653 3134-3135 231 ٨ MT14484T>C probe 13306-L14655 14484-14485 239 ٨ MT8344A>G probe 13309-L14658 8344-8343 reverse 247 ND5 (MTND5) probe 04316-L03672 14022-14023 251 MT3460G>A probe 13307-L14656 3460-3461 260 ATP6 (MTATP6) probe 13308-L14657 8993-8992 reverse 266 MT3243A>G probe 04904-L15252 3244-3243 reverse 286 ND1 (MTND1) probe 04290-L03646 4104-4105 294 ATP6 (MTATP6) probe 04303-L03659 9169-9170 307 ND6 (MTND6) probe 04318-L15459 14560-14561 313 ND2 (MTND2) probe 04291-L15460 4856-4857 321 COX3 (MTCO3) probe 04305-L03661 9326-9327 328 + CYTB (MTCYB) probe 04474-L15253 14825-14826 337 + ND2 (MTND2) probe 04475-L03648 5427-5428 350 COX3 (MTCO3) probe 04306-L03662 9915-9916 360 CYTB (MTCYB) probe 04308-L15254 15342-15343 369 MTTC/Y probe 04301-L03657 5822-5823 380 ND3 (MTND3) probe 04476-L15255 10171-10172 390 CYTB (MTCYB) probe 04309-L03665 15764-15765 400 COX1 (MTCO1) probe 04286-L15256 6022-6023 409 + ND4 (MTND4) probe 04311-L15257 10922-10923 418 ND1 (MTND1) probe 13312-L14661 3651-3652 427 ND5 (MTND5) probe 13313-L14662 13167-13168 ± 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 8994. * 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
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 ± 04293-L03649 RNR1 742-743 TAAATCACCACG-ATCAAAAGGGAC 349 nt 160 ± 13311-L15249 RNR1 1091-1092 ATACCCCACTAT-GCTTAGCCCTAA 598 nt 178 ± 04295-L03651 RNR2 1689-1690 ACCTAGCCCCAA-ACCCACTCCACC 1005 nt 202 ± 04296-L04299 RNR2 2694-2695 GACCTGCCCGTG-AAGAGGCGGGCA 440 nt 226 ± 04472-L03653 RNR2 3134-3135 CAAGAGAAATAA-GGCCTACTTCAC 109 nt 266 04904-L15252 3243A>G 3244-3243 rev. GATTACCGGGCC-CTGCCATCTTAA 217 nt 251 13307-L14656 3460G>A 3460-3461 CCTTCGCTGACA-CCATAAAACTCT 191 nt 418 13312-L14661 ND1 3651-3652 GCCGTTTACTCA-ATCCTCTGATCA 453 nt 286 04290-L03646 ND1 4104-4105 GACCCTACTTCT-AACCTCCCTGTT 752 nt 313 04291-L15460 ND2 4856-4857 ATCCGGCCTGCT-TCTTCTCACATG 571 nt 337 + 04475-L03648 ND2 5427-5428 CAGTTTGAACAT-ACAAAACCCACC 395 nt 369 04301-L03657 MTTC/Y 5822-5823 AAAATCACCTCG-GAGCTGGTAAAA 200 nt 400 04286-L15256 COX1 6022-6023 TATTCGAGCCGA-GCTGGGCCAGCC 1124 nt 148 04288-L03644 COX1 7146-7147 AAAATCCATTTC-ACTATCATATTC 503 nt 166 04298-L03654 COX2 7649-7650 GAAGAGCTTATC-ACCTTTCATGAT 263 nt 184 04299-L03655 COX2 7912-7913 CTGAACCTACGA-GTACACCGACTA 292 nt 211 04300-L03656 COX2 8204-8205 AACCACAGTTTC-ATGCCCATCGTC 139 nt 239 ٨ 13309-L14658 8344A>G 8344-8343 rev. AGAGGTGTTGGC-TCTCTTAATCTT 313 nt 220 04905-L03658 ATP6 8656-8657 AACCGACTAATC-ACCACCCAACAA 336 nt 260 13308-L14657 8993T 8993-8992 rev. GGCGTACGGCCA-GGGCTATTGGTT 177 nt 294 04303-L03659 ATP6 9169-9170 GCCTACGTTTTC-ACACTTCTAGTA 157 nt 321 04305-L03661 COX3 9326-9327 CTTCCACTCCAT-AACGCTCCTCAT 589 nt 350 04306-L03662 COX3 9915-9916 TTTGGCTTCGAA-GCCGCCGCCTGA 256 nt 380 04476-L15255 ND3 10171-10172 CCACCCCTTACG-AGTGCGGCTTCG 751 nt 409 + 04311-L15257 ND4 10922-10923 AGCTGTTCCCCA-ACCTTTTCCTCC 606 nt 154 04312-L03668 ND4 11528-11529 CTGACAAAACAC-ATAGCCTACCCC 250 nt 195 13305-L15250 11778G>A 11778-11779 CACTCACAGTCA-CATCATAATCCT 290 nt 171 04313-L03669 ND4 12068-12069 GAAAACACCCTC-ATGTTCATACAC 407 nt 190 04314-L03670 ND5 12475-12476 ACCTTTATTATC-AGTCTCTTCCCC 692 nt 427 13313-L14662 ND5 13167-13168 CAAACTCTAACA-CTATGCTTAGGC 855 nt 247 04316-L03672 ND5 14022-14023 TAGAAAAGCTAT-TACCTAAAACAA 462 nt 231 ٨ 13306-L14655 14484T>C 14484-14485 AAAGACAACCAC-CATTCCCCCTAA 76 nt 307 04318-L15459 ND6 14560-14561 AATAACACACCC-GACCACACCGCT 265 nt 328 + 04474-L15253 CYTB 14825-14826 ACCCCATCCAAC-ATCTCCGCATGA 517 nt 360 04308-L15254 CYTB 15342-15343 TCCACCTCCTAT-TCTTGCACGAAA 422 nt 390 04309-L03665 CYTB 15764-15765 ACCTGAATCGGA-GGACAACCAGTA 656 nt 136 13314-L14663 MT 16420-16421 TCCGTGAAATCA-ATATCCCGCACA 891 nt 142 ± 04293-L03649 RNR1 742-743 TAAATCACCACG-ATCAAAAGGGAC 349 nt 160 ± The mitochondrial DNA is circular and only 16.569 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 8994. + 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 www.mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P125 Mitochondria probemix Page 5 of 7
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
Implemented Changes compared to the previous product description versions. Version 18 31 October 2018 (55) - Warnings added to Table 1 and 2, 328 nt probe 04474-L15253, 337 nt probe 04475-L03648, and 409 nt probe 04311-L15257. - Warning adjusted in Table 1 and 2, 260 nt probe 13308-L14657. Version 17 26 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 16 18 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 15 23 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