Aging Aging

Mitochondrial DNA Mutations 25568°C † x min20 cycles 92–94°C* 10–30 s55–68°C** 30 s68°C † x min + 5 s per cycleFinal extension 68°C 15–20 min*Use lower denaturing temperatures and shorter times for longer products toavoid damaging the template.**Annealing temperature is dependent upon the oligonucleotide primers usedin the PCR reaction. For the amplification of a 13-kb product using L3200 andH16215, we use an annealing temperature of 58°C.† x depends on the length of product to be amplified; generally allow 1 min forevery 1 kb to be amplified.10. Place tubes in thermal cycler when there is 90 s remaining of the initial denaturationstep.11. When the program is complete, electrophorese 15–20 µL of the PCR productsthrough a 0.7% agarose gel at 65 V for 2–4 h (see Notes 3–5). The remainingproduct may be used for further analysis such as restriction enzyme digest orprimer-shift PCR to map the rearrangement (31).3.2. Single Cell PCR1. Histochemical staining: Tissue sections that have been stored at –85°C should beequilibrated at room temperature for 30 min, then removed from the air-tightcontainer and air-dried for a further 30 min prior to histochemical analysis. COXactivity is detected using incubation medium containing 4 mM 3,3'-diaminobenzidine tetrahydochloride and 100 µM cytochrome c. Each section isincubated with 100–200 µL of incubation medium at 37°C for up to 60 min in ahumid chamber. Any excess medium is rinsed using distilled water. The activityof SDH in the sections is detected using incubation medium containing 1.5 mMnitroblue tetrazolium, 130 mM sodium succinate, 0.2 mM phenazine methosulfateand 1 mM sodium azide. Each section is incubated at 37°C with 100–200 µL ofincubation medium, in a humid chamber for 30 min. Sections are rinsed in distilledwater to remove excess medium. COX-deficient cells are detected by adouble activity assay; sections are initially assayed for COX activity, as measuredby the production of a brown end product. After removal of the excessmedium, the sections are subsequently assayed for SDH activity. COX-deficientcells are easily identified as they do not produce the brown reaction product associatedwith COX activity, but do react for SDH activity, which gives a characteristicblue appearance (32). The neuronal cell harvesting is performed immediatelyafter histochemistry; however, muscle sections can be stored in 50% alcohol at4°C for several months.2. Micropipets: Micropipets of a specific diameter are produced from heat-sterilizedglass capillaries, using a micropipet puller in the double pull mode. A rangeof tip diameters (1–50 µm) can be made, depending on the size of the cells to be