General information
The SALSA MLPA
Probemix P125 Mitochondrial DNA is a
research use only (RUO) assay for the detection of deletions or duplications in the human mitochondrial DNA and the presence and allele fraction of six different point mutations as described in Tables 1 and 2 of the Product Description.
Mitochondrial DNA (mtDNA) differs from nuclear DNA. 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. The copy number of mitochondrial DNA is more than 100 fold higher as compared to genomic DNA. Most human cells contain over 200 copies of the mitochondrial genome.
The mutation rate in mtDNA is about 10 times higher than in nuclear DNA, probably due to an inferior repair system, the exposure to oxygen-free radicals generated by oxidative phosphorylation, and the lack of protective histones. The regions between nt 45-287 and between nt 16105-16348 are regarded as hypervariable. Mitochondrial DNA has no introns and hardly any intergenic regions. Most sequence changes will therefore affect a coding sequence. Transcription of mtDNA is polycistronic, which means that all genes encoded on the two (“heavy” and “light”) DNA strands are transcribed as two large precursor RNA strands. A deletion anywhere in the mitochondrial genome may affect the transcription or translation of other genes, even when their sequence is intact. As a result, deletions of various sizes can cause similar phenotypes. Inherited anomalies of mtDNA are always maternal as all mitochondria come from the ovum.
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. The most common deletion region is between positions 8469 and 13147. Diseases caused by deletions and point mutations in mtDNA are characterised by heteroplasmy: a mixture of wild-type and mutant mtDNA is present in one cell. A deletion present in 35% of the mitochondria results in a 35% signal reduction of the affected MLPA probes. Please note that identical deletions can cause different diseases depending on the tissue in which they occur.
Some tandem duplications of parts of the mitochondrial genome have also been described, as well as a number of frequent mitochondrial point mutations resulting in a myopathy, such as MERRF and MELAS.
More information is available at:
https://www.ncbi.nlm.nih.gov/books/NBK1224/ (Mitochondrial Disorders Overview).
https://www.ncbi.nlm.nih.gov/books/NBK1203/ (Mitochondrial DNA deletion syndromes).
https://www.ncbi.nlm.nih.gov/books/NBK1233/ (MELAS).
https://www.ncbi.nlm.nih.gov/books/NBK1520/ (MERRF).
https://www.ncbi.nlm.nih.gov/books/NBK1173/ (Mitochondrial DNA-Associated Leigh Syndrome and NARP)
https://www.ncbi.nlm.nih.gov/books/NBK1174/ (Leber Hereditary Optic Neuropathy)
Mitochondrial DNA mutation database:
https://www.mitomap.org/MITOMAP.
This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.
Probemix content
The SALSA MLPA Probemix P125-C1 Mitochondrial DNA contains 46 MLPA probes with amplification products between 124 and 463 nt. 34 probes are copy number probes.
Six probes are specific for six different frequent mutations which will only generate a signal when the mutation is present. For each of these six point mutations, a second probe is present that generates a signal only on the wildtype sequence of the mutation. Analysis of both the mutation specific probe and the associated wildtype probe provides an estimate of the allelic fraction of the point mutation. The six point mutations that are targeted are:
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m.3243A>G;
TL1 gene (Most frequent cause of MELAS)
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m.3460G>A;
ND1 gene (p.A52T; associated with Leber Hereditary Optic Neuropathy / LHON)
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m.8344A>G;
TK gene (Most frequent cause of MERRF)
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m.8993T>G;
ATP6 gene (associated with leigh syndrome and NARP)
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m.11778G>A;
ND4 gene (p.R340H; associated with Leber Hereditary Optic Neuropathy / LHON)
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m.14484T>C;
ND6 gene (p.M64V; associated with Leber Hereditary Optic Neuropathy / LHON)
This probemix contains four quality control fragments generating amplification products between 64 and 82 nt: the four DNA Quantity Fragments (Q-fragments). These will only be visible in reactions with extremely low amounts of sample DNA (< 1 ng). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at
www.mrcholland.com.
SALSA Binning DNA SD085
The SD085 Binning DNA provided with this probemix can be used for binning of the six mutation/SNP-specific probes (21433-L30223; 21436-L29939; 21440-L30473; 21443-L29946; 21446-L29949; 21452-L30472 ). SD085 Binning DNA is a mixture of genomic DNA from healthy individuals and a carefully titrated amount of plasmid DNA that contains the target sequence detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD085 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 signal(s), as for this purpose true mutation/SNP positive patient samples or cell lines should be used. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD085 Binning DNA product description, available online:
www.mrcholland.com.
This product is for research use only (RUO).
Sample DNA
Sample DNA developed for this product: