When looking at a mtDNA assay, the choice of target and/or control region will vary depending on the questions being asked by the investigator and by the origin of the DNA samples. One of the unique problems encountered when analyzing mtDNA is the presence of so many different reported DNA deletions. When designing the assay, it is important to bear in mind that mtDNA deletions accumulate with age, and that some deletion species are found more prevalently in some tissue types than others. What has been used as a reference target in one study may not necessarily work as a reference in a different tissue or patient population. The distribution of mtDNA deletions around the genome can be seen in Figure 11.2. The data have been taken from the MITOMAP web site (MITOMAP, 2005) and only deletions with reported breakpoints have been included. It can be clearly seen that the only region free from deletions is the D-loop. mtDNA species with deleted D-loop regions do not appear to persist in cells, most likely due to the removal of the regulatory regions preventing accumulation to detectable levels. It should be noted the D-loop is highly polymorphic and, although free from deletions, it is therefore not be the best choice as a reference region for an assay.
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