Aging Aging

Mitochondrial DNA Mutations 261X-100. This mix is then heated at 94°C for 5 min prior to adding 0.2 µL ofAmpliTaq ® DNA polymerase and commencing the PCR amplification asdescribed in Subheading 3.2., step 6.9. It is essential that preliminary control studies are done using samples containinga varying proportion of wild-type mtDNA and mtDNA 4977 , proportions determinedby Southern blotting. This ensures that the PCR reflects the two populationsof mtDNA in the original sample.10. The concentration of the template DNA affects the ratios of wild-type tomtDNA 4977 especially at very low concentrations (21).11. A control sample containing wild-type mtDNA and mtDNA 4977 of known proportionmust be run and quantitated on each PCR run.12. The template DNA must be of good quality, with an A 260 /A 280 ratio of no lessthan 1.8.13. Other restriction enzymes that linearize the mitochondrial genome (e.g., PvuII)can be used as an alternative to BamHI. Linearized DNA samples can be storedsuccessfully at –20°C for 2–3 d. Over longer periods of time, degradation of theDNA sample will lead to the appearance of additional, nonspecific bands followingPCR amplification.14. Serial dilution of DNA: It is essential that great care is taken at this stage toobtain an accurate dilution. This requires accurate pipetting and thorough mixingof each sample.15. DNA polymerase: We strongly recommend that AmpliTaq® DNA polymerase(Perkin–Elmer) is used. Cheaper alternatives do not give reliable amplification inour hands.16. Quantitation of PCR: When analyzing the gels of the PCR products, it is importantto link the IDV values to a local background setting.17. Primer pairs a and b are designed to amplify normal mtDNA from regions of thegenome that are rarely (primer pair a) and often (primer pair b) deleted (Fig. 3).They serve as good controls for effective cell lysis.18. PCR products generated by amplification with primer pairs c–h are specific tothe deletion found in that particular cell. Figure 2 shows the amplification of twodifferent deletions from individual COX-deficient muscle fibers, and highlightsthe presence of a single mutation at high levels in these fibers. These PCR productsencompass the deletion breakpoint, the sequence of which can be determinedby directly sequencing the primer-shift PCR product.19. If no visible products are amplified using any of the primer pairs, a secondaryPCR can be performed exactly as described using 1 µL of the initial PCR reactionas template.AcknowledgmentsThe financial support of the Northern Regional Health Authority, Researchinto Ageing, the Muscular Dystrophy Group of Great Britain, and the WellcomeTrust is acknowledged.