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SALSA MLPA KIT P125 Mitochondria
Mitochondrial DNA (mtDNA) differs from nuclear DNA in several ways. The complete mitochondrial genome is only 16,571 bp and is circular. Only a small number of genes needed for mitochondrial function are found within mtDNA; most are contained within nuclear. A deletion anywhere in the mitochondrial genome may affect transcription or translation of genes that were not affected by the deletion. Human cells contain on average 300-400 copies of the mitochondrial genome. Deletions of part of the mitochondrial genome have been described and are likely to cause several different diseases. Deletions vary in size (1.3-8 kb) and position within the mitochondrial genome; however the single most common site is between positions 8469 and 13147. Deletions are found in all tissues. These deletions have usually been detected by the formation of smaller fragments than expected in PCR reactions. Deletions of various sizes can produce similar phenotypes. Occasionally tandem duplications of DNA occur rather than deletions. Identical deletions have been identified in patients with different diseases such as Pearson syndrome, Kearns-Sayre Syndrome and Progressive external opthalmoplegia. In these diseases, mtDNA displays heteroplasmy, a mixture of wild-type and mutant mtDNA within a single cell. The ratio of mutant DNA to wild-type DNA becomes important in determining the phenotype in a mitochondrial disorder. In addition, the mitochondrial myopathies that are caused by gross rearrangements, several myopathies such as MERFF and MELAS are caused by point mutations. One MLPA probe is present in this probemix which is specific for the 3243A>G MELAS mutation. The MLPA technique has been used extensively to detect deletions and duplications of genomic sequences. Situations in which more than 50% of the mitochondrial genomes in diseased tissue contain a specific deletion will be rare. Instead of a 50% decrease or 50% increase in probe signal as is the case with genomic deletions / duplications, a much smaller change in probe signal is expected with MLPA probemixes for mitochondrial sequences. These will therefore probably only be detected when more than one probe sequence is located within the deleted area. Due to the large difference in copy number between genomic and mitochondrial sequences, it is not possible to use control probes for genomic sequences in this probemix. The probe mix included in this P125 MITO MLPA kit contains probes for 31 different sequences on the mitochondrial genome. We have not tested this probemix yet on patient DNA samples. We have no information yet on the minimum % of mutant mtDNA copies that can be detected with this probemix. Sensitivity of ordinary PCR assays will be higher. This multiplex MLPA assay may however have advantages in some cases. Deletions of probe recognition sequences will be apparent by a reduced relative peak area of the amplification product of that probe. However, mutations / polymorphisms very close to the probe ligation site may also result in a reduced relative peak area. Apparent deletions of a single exon will therefore always require confirmation by other methods. We have no information on which percentage of defects in these genes is caused by deletions / duplications of complete exons.
Full mix description (word)
Full mix description (pdf)
Last Update Probe mix description: Vs. 04; 16-03-2007 Last change in probe mix content: Lot 1105 (November 2005) Current Lot Number.: Lot 0606
IMPORTANT NOTICE: MLPA kits are sold by MRC-Holland 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 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)
References
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