Aging Aging

248 Taylor et al.(with a 3'→5' exonuclease proofreading activity) DNA polymerases to enablethe accurate and efficient amplification of PCR products up to 40 kb in size.1.2. Single-Cell PCRThe clonal expansion of mtDNA deletions within individual cells calls forsensitive PCR-based techniques to enable the identification and characterizationof these mtDNA mutations. Single-cell PCR on COX-deficient (biochemicallycompromised) cells highlights the focal nature of these mutational events,otherwise masked if analyzing DNA from tissue homogenates, and permits thecomparative study of mtDNA rearrangements in different cell types within thesame organ.This section describes both the histochemical analyzes required to identifythose cells that are biochemically affected as demonstrated by COX activity,and protocols for isolating total DNA from these single cells.1.3. Quantitative analysisof the 4977 Basepair Common Deletion in Single CellsThe PCR-based techniques described in the preceding section will detectany rearrangement between nt 8355 and nt 13832, but will not quantify theabsolute levels of mutant mtDNA (deletion). When searching for high levels ofdeleted mtDNA due to clonal expansion, competitive PCR (using three oligonucleotideprimers to simultaneously amplify both wild-type and mutantmtDNA) is a useful method for detecting and quantifying the common 4977-basepair deletion (mtDNA 4977 ) in single cells (23). The method described belowis essentially that of Sciacco and colleagues (21).1.4. Semiquantitative PCR of the 4977 Basepair CommonDeletion in Tissue HomogenatesAlthough the age-related accumulation of mtDNA deletions in various tissueshas been demonstrated, the absolute levels of rearrangement in total cellularDNA extracted from tissue homogenates are very low. Consequently,semiquantitative PCR-based methods have been described to permit the investigationof these accumulating mutations in different cell types. The protocolwe describe is based extensively on the method of Corral-Debrinski and colleagues(7,8), in which the PCR amplification of wild-type mtDNA andmtDNA 4977 from serially diluted DNA samples allows the estimation of theamount of deletion in a tissue.1.5. Primer-Shift PCRPrimer-shift PCR was first described by Ozawa et al. (28) as a means to finemap differentially deleted mtDNA species from total cellular DNA isolated fromtissue homogenates. The rationale is straightforward; using pairs of primers