Quantification of mtDNA deletions

When investigating mtDNA deletions there are two major approaches. The first will allow the measurement of the total mutation load in a sample, whereas the second is mtDNA deletion specific. The difference arises from the selection of amplicons. Both approaches require the selection of a control region which is not deleted in the cohort of samples under investigation. With the first approach the second amplicon is situated within the region deleted. This allows the measurement of wild-type mtDNA, enabling calculation of the total mutant load present by subtraction. This approach does not take into account whether this is from a single deletion or from multiple deletions which remove the wild-type amplicon region. The second approach is deletion specific. The second amplicon is located across the deletion breakpoint and so a product will only be amplified when a specific deletion is present. Both approaches can be used with DNA binding dyes or probe technologies. If selecting to use probe technology then having a primer on either side of the deletion with the probe sequence actually crossing the breakpoint will increase the reaction specificity to that single deletion.

Figure 11.2

Distribution of reported mtDNA deletions around the genome. Each single line represents a single DNA deletion species, the regions covered by the line being those removed by the deletion. Data taken from the MITOMAP web site. Only deletions with reported breakpoints are illustrated. Numbers show the base positions relative to the revised Cambridge Reference Sequence (rCRS). The bias towards the major arc can clearly be seen.

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