Quantitative Southern blot analysis can be used for the quantitation of oxidative damage to mtDNA. This method is based on the detection of strand breaks within linearized mtDNA. These strand breaks can be generated by oxidative stress or by treatment of DNA containing oxidative base lesions with either FPG (recognizes oxidized purines) or E. coli endonuclease III (EndoIII, recognizes oxidized pyrimidines) followed by alkaline agarose gel electrophoresis. Under the alkaline conditions, the two mtDNA strands separate and single strand breaks occur at abasic sites created by the activity of FPG or EndoIII, which results in a decreased hybridization signal from the damaged DNA (LeDoux et al., 1999).
An alternative approach for the detection of mtDNA damage was developed by Bennett van Houten. This method, QPCR, is based on the ability of the lesions present in mtDNA to block the progression of thermostable DNA polymerase on a template, resulting in a decrease of DNA amplification in the damaged template when compared to undamaged DNA (Yakes and Van Houten, 1997). The QPCR actually measures the fraction of undamaged template, which decreases with increased number of lesions.
The successful outcome of experiments with both quantitative Southern blot and QPCR is heavily dependent upon the ability to accurately measure the amount of DNA used. Spectrophotometry methods (A260) appear to be inappropriate for this purpose because of the intrinsic difficulties associated with controlling the quantity and spectrum of contaminants in DNA preparations. Fluorescence-based methods (PicoGreen and Hoechst33258 dyes), unlike spectrophotometry techniques, show little sensitivity to such contaminants as proteins, single-stranded DNA, RNA, and so on, which are common to genomic DNA preparations and therefore are deemed methods of choice. Also, when using QPCR, one has control for changes in the mtDNA copy number. Indeed, reduction in mtDNA copy number will manifest itself as DNA damage because of the reduction in the number of amplifiable mtDNA genomes in the template. To control for this problem, amplification of a short (about 300bp) fragment of the gene under the study is performed. The rationale is that encountering DNA damage in such a short fragment is an event with a very low probability, and therefore profiles of amplification of such a fragment should be essentially identical between damaged and undamaged DNA. Therefore, variations in the degree of amplification are assumed to be the result of fluctuations in mtDNA copy number, and the results of small fragment amplification are used for the normalization of the data obtained for the large (15 kb) mtDNA fragments.
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