MtDna Sequencing

The total burden of mtDNA point mutations in tissues can be quantified using mtDNA sequencing. This approach measures consequences of oxidative stress and other mutagenic insults on the integrity of mtDNA. The protocol includes PCR amplification of specific DNA

regions, cloning of the PCR fragments, and sequencing either resulting plasmid DNA or PCR template amplified from it (Simon et al., 2004). The high sensitivity of this technique raises several technical concerns. The first is that PCR errors might be misinterpreted as somatic mtDNA mutations. The use of a high-fidelity PCR system limits this possibility. However, the fidelity of DNA polymerase can be affected by experimental conditions, and therefore the spectrum and the level of contaminants in DNA preparations can affect the level of PCR-induced mutations. The fidelity of this approach was experimentally tested by amplifying a single clone followed by a repeat cloning step. The lack of identification of any mutations out of 107 clones and nearly 98,000 base pairs of sequencing demonstrates the high fidelity of this method. A second concern is that oxidative damage to the original template DNA might lead to the induction of mutations due to replication errors in the early precloning PCR cycles. This possibility is not ruled out by the preceding control study, since cloned DNA would not necessarily have the same oxidative damage as the original DNA. However, a control study failed to detect a significant difference in mutation frequencies when the template DNA was exposed ex vivo to high levels of oxidative stress (Lin et al., 2002). The third concern is that it remains possible that some other form of template damage that differs between DNA derived from different brain regions or from subjects of different ages may lead to PCR errors. The fourth concern is inadvertent amplification of nuclear pseudogenes. There are 612 independent integrations of mtDNA sequences evenly distributed over the nuclear genome. Our observations indicate that nuclear insertions of mitochondrial sequences occurred independently in human and mouse lineages after their separation from a common ancestor (see Figure 41.3).

Interestingly, no integration of the D-loop region into the nuclear DNA has occurred in either human or mouse genomes, which makes studies focused on mutations in this region free of potential artifacts introduced by amplification of pseudogenes. The interference from nuclear pseudogenes is generally pertinent only to large-scale studies because mtDNA present in the cell in a great molar excels over the nuclear DNA. Additionally, most regions of homology are relatively small and will not interfere if the experimental design involves amplification and sequencing of large mitochondrial fragments. Finally, because of the accumulation of mutations in nuclear pseudogenes, it is often possible to design primers that would specifically amplify mtDNA.

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