Analysis of mitochondrial DNA point mutation frequency with age by Random Mutation Capture (RMC).

<p>(<b>A</b>) Schematic diagram describing the RMC methodology. (<b>i</b>) Schematic diagram of the structure of the human colorectal crypt. (<b>ii</b>) Schematic diagram showing mtDNA isolation. Colonoscopic biopsies are homogenized and the mitochondrial fraction isolated by differential centrifugation. MtDNA is then prepared by phenol/chloroform extraction and quantified using real-time PCR (standard curve method). (<b>iii</b>) MtDNA is digested for 10 hours with <i>Taq1α</i>. PCR is then carried out over the restriction site. Only molecules with mutations in the restriction site are able to successfully amplify. (<b>iv</b>) Agarose gel showing PCR products from a typical RMC run. Each reaction contained ∼10000 target base pairs. 488 base pair bands show amplified mutated molecules (wells 4,6,13,16 and 20). The wild-type control well (Wt) shows complete digestion of wild-type DNA following PCR. (<b>v</b>) Example electropherograms showing mutations (asterisks) within the restriction site (highlighted in blue). (<b>B</b>) Frequency of all RMC detected mtDNA mutations in human colorectal mucosa (n = 207). There was no correlation between mtDNA mutation frequency and age (Pearson correlation = 0.127 (P = 0.07)). (<b>C</b>) Data from (<b>B</b>) presented on a log 10 scale to show the spread of the data. Note that the zero values cannot be displayed in this way therefore n = 175. (<b>D</b>) Frequency of all mtDNA mutations detected in human colonic mucosa, grouped by decade. Subjects were grouped as follows, 17–26 (n = 12), 27–36 (n = 19), 37–46 (n = 58), 47–56 (n = 51), 57–66 (n = 43), 67–77 (n = 23). There were no significant differences between any of the groups (P = 0.343, One Way ANOVA).