Aging Aging

Mitochondrial DNA Mutations 249designed to amplify both (H) and (L) strands and relatively short extension times,PCR products are obtained only if a deletion is present, thereby shifting thePCR primers closer together, facilitating amplification. These PCR products willcontain the specific deletion breakpoint, and as such can either be cloned orsequenced directly to map the precise location. Furthermore, because this techniqueexcludes nonspecific PCR amplification due to the misannealing of PCRprimers, it amplifies only deleted mtDNA species. Consequently, primer-shiftPCR has been used to characterize the multiple mtDNA associations observedin patients with inclusion body myositis (29,30) and demonstrate the clonal expansionof mtDNA deletions in COX-deficient skeletal muscle fibers of patientswith adPEO (22). This study highlights the power of this technique to screen formtDNA deletions associated with disease or the aging process in DNA isolatedfrom a single cell. The protocol described in the following sections describesthe use of primer-shift PCR to investigate the presence of mtDNA deletions inDNA isolated from single muscle fibers or individual neurons. The same methodis easily applied to the characterization of deleted mtDNA species in total cellularDNA isolated from a tissue homogenate.2. Materials2.1. Long-range PCR of mtDNA1. Expand Long Template PCR system (Boehringer Mannheim): This is suppliedwith the enzyme mix and three buffers. Reaction buffer 3 is suitable for mostapplications as it contains dimethyl sulfoxide (DMSO) (20% [v/v]) which preventsDNA depurination and intrastrand secondary structure formation. Theenzyme can be stored at –20°C for approx 3 mo. Reaction buffer 3 should bechecked for the appearance of crystals that may have precipitated before use.2. Deoxynucleoside triphosphates (dNTPs): Separate 10 mM working solutions ofdATP, dCTP, dGTP, and dTTP are recommended. These are made from 100 mMlithium salt dNTP stocks purchased from Boehringer Mannheim. Store at –20°C.3. Bovine serum albumin (BSA): Although not essential for the reaction, the additionof BSA (200 µg/mL final concentration) may increase the efficiency of thelong template amplification. A 10 mg/mL stock solution of BSA that is oftensupplied with restriction endonucleases can be diluted to a 1 mg/mL workingsolution for this purpose, and stored at –20°C.4. Oligonucleotide primers: Any primers designed to amplify mtDNA may be used;however, successful amplification may require testing various combinations ofprimers and annealing temperatures. Increasing the annealing temperature andreducing primer concentrations may help to reduce any non-specific PCR amplification.We find that short primers (20–24 bases long) give good results, whereaslonger primers (>30 bases) do not. Interestingly, this inefficiency of amplificationmay be overcome by using a short primer in combination with a long primer.For much of our routine screening, we amplify a 13-kb fragment of the mitochon-