Single-Step Method for the Determination of the Amount of the Common Deletion in Mitochondrial DNA

Abstract

A fluorescent-based quantitative PCR assay to measure the percentage of the common deletion in rat mitochondria is reported. The amount of the common deletion is measured relative to the amount of total mitochondrial (mt)DNA. The use of a heterologous competitor construct allows the reaction to be monitored to ensure that exponential accumulation of products occurs. The use of fluorescence-labeled primers provides a safe and sensitive means to quantify products without any adjustment for size. This assay will allow the quantitative determination of the common deletion with one dilution range.

Microsatellite instability, mitochondrial DNA large deletions, and mitochondrial DNA mutations in gastric carcinoma

“…In contrast to the other series (Burgart et al, 1995;Habano et al, 2000), we detected ⌬4977 bp mtDNA mutations in gastric carcinomas (Máximo et al, 1999). This may be due to differences in the technical procedure, since it is known that the detection of low levels of the ⌬4977 bp mtDNA requires the specific and sensitive techniques we have been using (Máximo et al, 1998(Máximo et al, , 1999Mü ller-Hö cker et al, 1998;Ahmed et al, 1999;Lewis et al, 2000).…”

Torsion‐induced injury in rat testes does not affect mitochondrial respiration or the accumulation of mitochondrial mutations

“…We determined the extent of the common deletion in the testis samples using a quantitative assay (Ahmed et al ., 1999). Statistically significant differences were not seen between the control and the torsioned testes, and both showed similar levels of deletions to those in the untreated control and the control groups (Fig.…”

“…The values of the 4834 bp deletion measured in all the animals ranged between 0.007 to 0.072% of the total mitochondrial genome. These values are similar to those that we have previously measured for other tissues in similarly aged rats that have not undergone any experimental treatment (Ahmed et al ., 1999; unpublished data). It is evident that, while the morphological data indicate progressive damage over time, no changes could be detected in terms of the accumulation of the common deletion.…”

“…All reactions amplifying control or deletion sequences from a single sample were performed simultaneously. Mixes that contained a mix of the cloned control and deletion fragments at concentrations of 0.1, 1 and 10% deletion were used regularly as positive controls (Ahmed et al ., 1999).…”

“…We determined the extent of the common deletion in the testis samples using a quantitative assay (Ahmed et al, 1999). Statistically signi®cant differences were not seen between the…”