Aging Aging

Mitochondrial DNA Mutations 247F.P.O.Fig. 1. 13 kb long-range PCR of mtDNA using primers L3200 and H16215.M, λ-HindIII DNA markers; C, control subject showing a single wild-type band;lanes 1–5, subjects demonstrating different mtDNA rearrangements.mtDNA is high compared to wild-type. Because of the number of polymerasechain reaction (PCR)-based techniques available, we describe these protocolsin two chapters. In this, the first chapter, we discuss the methods used to investigatemtDNA deletions, whereas the second chapter (Chapter 20) focuses onthe analysis of mtDNA nucleotide substitutions.1.1. Long-Range PCR of mtDNALong-range PCR is now commonly used as an initial screen for the presenceof large-scale rearrangements (deletions or duplications) of mtDNA (24). Inthis respect, it has many advantages over Southern Blotting. Low levels of rearrangedmtDNA species can be detected from small amounts of DNA templatewithin one day, whereas Southern blotting requires 2–3 µg of total DNA, takesa number of days to complete, and has a detection threshold of about 5% mutantmtDNA. For these reasons long-range PCR has been used to study the accumulationof age-related deletions in muscle and other tissues (25,26) as well ascharacterizing pathogenic rearrangements in blood (27) and muscle (Fig. 1).Although there are a number of commercially available kits for long-rangePCR, we and others favor the Expand Long Template PCR system producedby Boehringer Mannheim. This uses a mixture of thermostable Taq and Pwo